Antibody molecules

ABSTRACT

The present invention provides murine monoclonal antibody molecules that bind to Merkel cell carcinoma virus (MCV) polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a divisional of U.S. patent application Ser.No. 12/808,042 filed Jan. 21, 2011 which is a U.S. National Phase ofInternational Patent Application No. PCT/US08/86895, filed Dec. 15,2008. This patent application claims the benefit of U.S. ProvisionalPatent Application No. 61/013,772 filed Dec. 14, 2007. The contents ofeach of these prior applications are incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under Grant Number NIHR33CA120726 awarded by the National Institutes of Health. The Governmenthas certain rights in this invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 107,991 Byte ASCII (Text) file named“713923_ST25 (2).TXT” created on Oct. 9, 2013.

BRIEF SUMMARY OF THE INVENTION

The present invention provides isolated or substantially purifiedpolypeptides, nucleic acids, and virus-like particles (VLPs) derivedfrom a Merkel cell carcinoma virus (MCV), which is a newly-discoveredvirus. The invention further provides monoclonal antibody molecules thatbind to MCV polypeptides. The invention further provides diagnostic,prophylactic, and therapeutic methods relating to the identification,prevention, and treatment of MCV-related diseases. These aspects, andother inventive features, will be apparent upon reading the followingdetailed description in conjunction with the accompanying figures andsequence listing.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1. A) Merkel cell carcinoma (MCC). MCC is an aggressive small,round-cell skin cancer of Merkel mechanoreceptor cells with highermortality rates than most other skin cancers (Left panel: H&E, Rightpanel: cytokeratin 20 staining, 40×). B) 3′-RACE mapping of MCV Tantigen-human PTPRG fusion transcript. The viral transcript discoveredby DTS in MCC347 was extended by 3′-RACE. Three mRNA sequences wereisolated, two of which terminate in human PTPRG intron 1 on Chromosome3p14. The two viral-human transcripts were generated by read-through ofa weak polyadenylation signal in the viral T antigen gene.

FIG. 2. Neighbor-joining trees for MCV putative large T (T-1, see FIG.3), small T (T-2), VP1 and VP2 proteins. The four known humanpolyomaviruses (BK, JC, KI and WU) cluster together in the SV-40subgroup while MCV is most closely related to MuPyV subgroup viruses.Both subgroups are distinct from the avian polyomavirus subgroup.

FIG. 3. Northern and 3′-, 5′-RACE mapping of MCV T antigen transcriptsin MCC tumors and during expression in 293 cells. Genomic T antigenfragments from MCV350 and 339 were expressed in pcDNA vector in 293cells. The left panel shows northern blotting of the 293 cell RNAsallowing individual transcripts to be assigned to four distinct bands.Higher molecular weight bands in this northern blot exceed the genomefragment size and are artifactually produced from vector expression(note difference sizes between pcDNA339 and pcDNA350 result from a 201nt. deletion in MCV339 T antigen sequence). T-1 is similar topolyomavirus large T antigens and possesses highly conserved crl, DnaJ(HPDKGG), LXCXE, origin binding domain (OBD) and helicase/ATPasefeatures. Stop codons and deletions prevent full-length large T proteinexpression in tumor derived viruses. T-2 possesses a large intronicsequence resulting in a protein similar to small T antigens from otherpolyomaviruses. An additional 0.8 kbase band is expressed in 293 cellsbut no corresponding tumor RACE product was isolated.

FIG. 4. A) Clonal MCV integration in MCC tumors detected by directSouthern hybridization. DNA digests with BamHI (left) or EcoRI (right)and Southern blotted with seven MCV DNA probes reveals different bandingpatterns in each tumor, including >5.4 kbase bands. Open arrow shows theexpected position for MCV episomal or concatenated-integrated genome(5.4 kbase) with corresponding bands present in tumors MCC344 and 350.Tumors MCC339, 347/348, and 349 have different band sizes and doubletbands (closed arrows) consistent with genomic monoclonal integration.MCC352 has both prominent episomal/concatenated bands (EcoRI), andhigher and lower molecular weight integration bands (BamHI). TumorsMCC337, 343 and 346 have no MCV DNA detected by Southern blotting (bandsat 1.5 kbase (kidney) and 1.2 kbase (MCC346) are artifacts). B) Viraland cellular monoclonality in MCC347. Tumor MCC347 and its metastasisMCC348 were digested with SacI and NheI, and Southern blotted withunique human flanking sequence probe (Chr3 (red), left panel) or viralprobes (LT1 and LT2 (yellow), right panel). The wild-type human alleleis present in all samples at 3.1 kbase (left panel). The MCC tumors,however, have an additional 3.9 kbase allelic band from MCV insertioninto 3p14. Probing with for MCV T antigen sequence (right panel)generates an identical band.

FIG. 5. Representative results of MCC and control tissue PCR-Southernblotting. (Top panel) Agarose gel of amplification products from 16randomized control and MCC tissue samples using LT1 primers (seeSupplementary Table 4) (Bottom panel). Specific hybridization of PCRproducts to a (α32P) dCTP-labeled M1-M2 internal probe (seeSupplementary Table 4) after transfer of DNA to nitrocellulose membrane.Sample identities are as follows: MCC tissue samples in lanes 1(MCC346), 5 (MCC348), 6 (MCC344), 9 (MCC339), and 15 (MCC343); negativecontrol (H2O) samples in lanes 2, 10 and 11; and surgical control tissuesamples in lanes 3, 4, 7, 8, 12, 13, 14 and 16. Weak signal in lane 13(control gall bladder tissue) is positive only after Southern blottingof the PCR product compared to robust PCR amplification for MCC348, 344,339 tissues. MCC346 and 343 are negative.

FIG. 6. BamHI-EcoRI double-digestion and Southern hybridization of theMCV T antigen locus. BamHI-EcoRI should generate a single ˜1.5-1.7 kbasefragment from T antigen (see FIG. 72A) unless genomic integration ordeletion occurs in this region. Marked variation in band sizes (MCC339,344, 345, 347, 348) are consistent with either human genomicintegrations or deletions within T antigen locus. Open arrow indicatesexpected BamHI-EcoRI viral fragment (1.7 kbase) for MCV350 and closedarrow, expected T antigen fragment (1.5 kbase) for MCV 339.

FIG. 7. MCV genome diagram showing large T, small T, VP1, andoverlapping VP2 and VP3 genes and DTS1 and DTS2. The former was used toidentify MCV and latter is a spliced transcript having low polyomavirushomology.

FIG. 8 graphically illustrates the location of the ORFs within thegenome of MCV 350.

FIG. 9 presents data showing CM2B4 mAb is specific for MCV LT. (A) A MCVT antigen-EGFP fusion protein colocalizes with CM2B4 staining in 293cells transfected with pMCV TAg-EGFP or pEGFP, an empty vector. (B)CM2B4 does not react with T antigens of human polyomaviruses belongingto SV40 subgroup. Constructs encoding LT genes for JCV, BKV and MCV wereexpressed in 293 cells and stained with CM2B4 or PAb416 antibodies.PAb416 cross reacts with JCV and BKV LT proteins but not with MCV LT.(C) Immunoblotting for expressed polyomavirus LT with PAb416 and CM2B4antibodies in cell lysates described in (B). CM2B4 recognizes an MCV 120kDa (LT) protein and a shorter 60 kDa T antigen isoform. Nocross-reactivity is apparent for PAb416 with MCV LT or CM2B4 withSV40-group polyomavirus proteins. (D) Brain tissue with progressivemultifocal leukoenchephalopathy show JCV infection of oligodendroglialcells by JCV specific in situ hybridization (left panel), and CM2B4shows no reactivity to JCV antigens (right panel). (E) Detection oftruncated LT protein by CM2B4 in MCV positive MKL-1 cell line. Proteinsfrom MKL-1 cells and MCV negative UISO, MCC13 and MCC26 cells wereimmunoblotted with CM2B4. LT antigen bands are only present in MKL-1cells.

FIG. 10 presents data showing specific and uniform expression of MCV LTprotein in MCC. (A) Uniform expression of MCV LT in MKL-1 cell line.Representative sections showing MCV LT and CK20 protein expression inMKL-1 and UISO cells. (B) MCC tissue specific expression of LT protein.Representative sequential sections from MCC showing histologicalphenylype (H&E) and immunostaining for MCV LT (CM2B4) and CK20 proteins.Expression of MCV LT protein expression is precisely localized innucleus of MCC cells but not in surrounding tissues including theepidermis, adnexal epithelium (arrow), endothelial cells, or dermalfibroblasts.

FIG. 11 presents data demonstrating the construction of MCV VLPs.

FIG. 12 presents data demonstrating the construction of MCV VLPs. Thetop panel shows an anti-MCV Western blot of 293TT cells aftertransfection with the VP1 expression construct shown, together with anappropriate VP2 expression construct. In the far right lane of theWestern, 5-fold more cell lysate was applied to the gel. The bottompanel shows a SYPRO Ruby-stained SDS-PAGE gel analysis of Optiprepgradients used to purify VLPs out of cell lysates. For MPyV and MCV399,2.5 μl each of fractions 6-9 was loaded onto the gel. For MCV350, 12.5μl each of fractions 6-9 was loaded. Fractions were screened for thepresence of encapsidated DNA using Picogreen reagent.

FIG. 13 presents data showing the determination of serum sample workingdilution for competitive ELISA for MCV VLP. This figure represents asingle experiment with one serum sample. Working dilution was estimatedas lowest dilution at which OD was greater than 1.

FIG. 14 presents data showing examples of VP1 peptides screen (A-B) ofMCV-positive sera (A) and negative sample (B) diluted 1:500.

FIG. 15 presents data showing the extent of positive reaction to MCV VLPELISA among Langerhan's cell histiocytosis patients of various agegroups.

FIG. 16 presents data showing the results of the reactivity (ELISA) of12 sera samples to BKV VLPs.

FIG. 17 presents data showing competitive ELISA with BK- and MCV-VLPs.This figure demonstrates results of the typical experiment withMCV-positive serum.

FIG. 18 presents data showing the seroreactivity of 12 serum samples toHPV and CRPV VLPs. A—HPV VLP, B—CRPV VLP ELISA.

FIG. 19 presents the results of ELISA assays for MCV VLPs in variouscohorts (confirmed MCC patients positive with MCV, lupus patients,Langerhan's cell histiocytosis patients, blood from commercial sources,and serum form blood donors).

FIG. 20 presents data showing the OD values of 20 confirmed MCC patientspositive with MCV (left) and from blood donors.

FIG. 21 presents data showing validation of a MCV neutralization assay.The

line shows an MCV neutralization curve for IgG purified out of thepooled human serum using protein G resin (starting concentration 1mg/ml). The

line shows results using serum after passage over protein G resin. The Xand O points display, respectively a general lack of neutralization ofan MPyV reporter vector by the pooled human serum and completeneutralization of the MPyV vector by MPyV-specific rabbit serum.

-   -   FIGS. 22A-22K present sequences discussed herein.

DETAILED DESCRIPTION OF THE INVENTION

Within the context of the present invention, a nucleic acid sequence isconsidered to be “selectively hybridizable” to a reference nucleic acidsequence if the two sequences specifically hybridize to one anotherunder moderate to high stringency hybridization and wash conditions.Hybridization conditions are based on the melting temperature (T_(m)) ofthe nucleic acid binding complex or probe. For example, “maximumstringency” typically occurs at about T_(m)−5° C. (5° C. below the T_(m)of the probe); “high stringency” at about 5-10° C. below the T_(m);“intermediate stringency” at about 10-20° C. below the T_(m) of theprobe; and “low stringency” at about 20-25° C. below the T_(m).Functionally, maximum stringency conditions may be used to identifysequences having strict identity or near-strict identity with thehybridization probe; while high stringency conditions are used toidentify sequences having about 80% or more sequence identity with theprobe. This is especially true for polynucleotides having a minimum offrom about 18-22 nucleic acids, but those of ordinary skill in the artare also able to apply these principals to larger or smallerpolynucleotides.

Moderate and high stringency hybridization conditions are well known inthe art (see, for example, Sambrook et al. Molecular Cloning: ALaboratory Manual (Second Edition), Cold Spring Harbor Press, Plainview,N.Y., 1989, especially chapters 9 and 11; and Ausubel F M et al. CurrentProtocols in Molecular Biology, John Wiley & Sons, New York, N.Y.,1993). An example of high stringency conditions includes hybridizationat about 42° C. in 50% formamide, 5×SSC, 5×Denhardt's solution, 0.5% SDSand 100 μg/ml denatured carrier DNA followed by washing two times in2×SSC and 0.5% SDS at room temperature and two additional times in0.1×SSC and 0.5% SDS at 42° C.

Furthermore, within the context of the present invention, it will beunderstood that a protein (such as an antibody molecule, but alsoapplicable to other proteins (e.g., in the context of binding otherproteins or DNA)) “binds selectively” to a target (e.g., an antigen,target DNA, etc.) if it binds to the target to a significantly higherdegree than it binds to another substance (e.g., another protein or anon-specific DNA sequence). Those of ordinary skill will understand thisconcept as being a hallmark of immunoglobulin-antigen interaction or theinteraction between a DNA-binding protein and its target consensussequence.

One aspect of the present invention relates to proteins and nucleicacids (DNAs or RNAs) derived from the newly-identified MCV. In thisrespect, one embodiment of the present invention provides an isolated orsubstantially purified nucleic acid molecule comprising at least about 6contiguous nucleotides of a MCV genomic DNA sequence. The completegenomic DNA sequence of MCVs from four tissue sources (MCV 350 (SEQ IDNO:1), MCV 339 (SEQ ID NO:2), MCV 352 (SEQ ID NO:3) and MCV MKL1 (SEQ IDNO:4) are set forth in the Sequence Listing. Amino acid sequences forthe VP1, VP2, VP3, T-1, T-2, T-3, T-4, and T-5 proteins, as well astheir coding DNA sequences, are set forth in the Sequence Listing as SEQID NOs:5-20. DNA sequences for MCVs isolated from small cell lungcarcinomas are set forth as SEQ ID NOs:21-45 in the Sequence Listing.Furthermore, possible ORFs of the MCV 350 genome (SEQ ID NO:1) aredisplayed in FIGS. 7 and 8. With reference to the genome itself (SEQ IDNO:1), these ORFs are further identified in the following table A:

TABLE A Sense Strand Antisense Strand  124 to 756 length = 633 C 5386 to5294 length = 93  330 to 410 length = 81 C 5324 to 5064 length = 261 362 to 469 length = 108 C 5266 to 5141 length = 126  470 to 595 length= 126 C 5148 to 4393 length = 756  591 to 695 length = 105 C 5068 to4973 length = 96  623 to 715 length = 93 C 4846 to 4760 length = 87  716to 865 length = 150 C 4718 to 4620 length = 99  780 to 860 length = 81 C4433 to 3156 length = 1278  835 to 1536 length = 702 C 4429 to 4250length = 180  861 to 1403 length = 543 C 4249 to 4094 length = 156  893to 1132 length = 240 C 4182 to 4090 length = 93 1319 to 1501 length =183 C 3975 to 3874 length = 102 1404 to 3080 length = 1677 C 3742 to3638 length = 105 1537 to 1626 length = 90 C 3222 to 3016 length = 2071834 to 1959 length = 126 C 3205 to 3122 length = 84 2023 to 2175 length= 153 C 3125 to 2949 length = 177 2132 to 2227 length = 96 C 3073 to2993 length = 81 2237 to 2323 length = 87 C 3006 to 2914 length = 932275 to 2364 length = 90 C 2965 to 2834 length = 132 2365 to 2445 length= 81 C 2862 to 2749 length = 114 2414 to 2491 length = 78 C 2735 to 2652length = 84 2753 to 2881 length = 129 C 2703 to 2530 length = 174 2818to 2973 length = 156 C 2623 to 2354 length = 270 2986 to 3129 length =144 C 2529 to 2410 length = 120 3047 to 3133 length = 87 C 2441 to 2361length = 81 3134 to 3319 length = 186 C 2349 to 2233 length = 117 3162to 3311 length = 150 C 2284 to 2135 length = 150 3283 to 3381 length =99 C 2232 to 2128 length = 105 3382 to 3465 length = 84 C 2114 to 2019length = 96 3450 to 3584 length = 135 C 2014 to 1919 length = 96 3523 to3657 length = 135 C 1998 to 1906 length = 93 3642 to 3770 length = 129 C1905 to 1804 length = 102 3802 to 3885 length = 84 C 1873 to 1640 length= 234 3863 to 4012 length = 150 C 1667 to 1533 length = 135 4006 to 4128length = 123 C 1588 to 1400 length = 189 4017 to 4097 length = 81 C 1392to 1222 length = 171 4195 to 4443 length = 249 C 1387 to 857 length =531 4209 to 4301 length = 93 C 1274 to 1182 length = 93 4370 to 4567length = 198 C 1221 to 922 length = 300 4467 to 4544 length = 78 C 911to 825 length = 87 4545 to 4646 length = 102 C 856 to 761 length = 964589 to 4873 length = 285 C 760 to 677 length = 84 4737 to 4850 length =114 C 725 to 645 length = 81 4935 to 5030 length = 96 C 711 to 601length = 111 4946 to 5134 length = 189 C 652 to 560 length = 93 5131 to5292 length = 162 C 641 to 474 length = 168 5205 to 5336 length = 132 C591 to 466 length = 126 5216 to 5320 length = 105 C 473 to 318 length =156 C 465 to 310 length = 156 C 309 to 208 length = 102 C 263 to 147length = 117 C 256 to 134 length = 123 C 201 to 67 length = 135 C 146 to63 length = 84 C 85 to 8 length = 78

Exemplary MCV genomic DNA sequences from which the inventive nucleicacid can be derived from include, but are not limited to, SEQ ID NOs:1-5, 7, 9, 11, 13, 15, 17, 19, and 21-45. Thus, for example, theinventive DNA can include from about 10 to about 20 contiguousnucleotides of such MCV genomic DNA sequences, the majority ofcontiguous nucleotides of such MCV genomic DNA sequences, substantiallyall of such MCV genomic DNA sequences, or even including the completesequence set forth in SEQ ID NOs: 1-5, 7, 9, 11, 13, 15, 17, 19, or21-45 or other MCV genomic DNA sequence. It will be understood that theinvention also includes the complement of such sequences. Furthermore,the invention also includes a nucleic acid that hybridizes under highstringency conditions to such sequences.

As minor differences in sequence are tolerated, so long as they do notimpede the function of the nucleic acids, the invention further providesan isolated or substantially purified nucleic acid molecule consistingessentially of at least about 6 contiguous nucleotides of a MCV genomicDNA sequence, such as those discussed herein. Thus, for example, theinventive DNA can consist essentially of from about 10 to about 20contiguous nucleotides of such MCV genomic DNA sequences, the majorityof contiguous nucleotides of such MCV genomic DNA sequences,substantially all of such MCV genomic DNA sequences, or even consistingessentially of the complete sequence set forth in SEQ ID NOs: 1-5, 7, 9,11, 13, 15, 17, 19, or 21-45 or other MCV genomic DNA sequence. It willbe understood that the invention also includes the complement of suchsequences as well a nucleic acid that hybridizes under high stringencyconditions to such sequences.

In one respect, the inventive isolated or substantially purified nucleicacids can be employed as probes, for example in diagnostic assays foridentifying MCV. In this respect, while it has been recited that theinventive nucleic acid can comprise at least about 6 contiguous nucleicacids from an MCV genomic sequence, somewhat shorter contiguous residuescan be permitted, if the molecule is nonetheless capable of hybridizingunder high stringency to MCV genomic DNA or its complement. Moreover,the length of the probe can vary to be as long as useful for theassay-in-question. Thus, the probe can comprise about 12-15 nucleotides,or can comprise longer sequences, such as about 20 or about 25nucleotides, if desired. Of course, a probe also can have sequencesother than MCV sequences, such as restriction endonuclease consensusrecognition sequences to facilitate cloning.

In other respects, the inventive the inventive isolated or substantiallypurified nucleic acids can be employed as agents to interfere with viralreplication. Thus, the inventive nucleic acid can be or comprise anoligodeoxynucleotide, siRNA molecule, or other suitable type ofpolynucleic acid. Such molecules can include standard modifications tostructure or employed modified sequences/nucleotides (e.g., generationof hairpins, use of triphosphate modified dNTPs, etc.) to enhanceactivity or stability of such molecules.

In yet further aspects, certain of the inventive nucleic acids encodeMCV proteins and polypeptides, and the invention provides such encodingnucleic acids, as well as nucleic acids which complement such or whichhybridize to such under high stringency. Thus, for example, theinventive isolated or substantially purified nucleic acid molecule canencode all or a portion of an MCV polypeptide. Examples of some suchpolypeptides include SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, and anyopen reading frame (ORF) of SEQ ID NOs: 1-4. ORFs of SEQ ID NO:1 arerepresented in table A, but putative ORFs of SEQ ID NOs: 2-4 can bededuced by those of ordinary skill in the art. Typically, the nucleicacid will encode a polypeptide that includes at least about 4 or about 5contiguous nucleic acid residues of an MCV protein, and oftensubstantially more contiguous nucleic acids from such proteins (e.g., atleast about 10 contiguous nucleic acids or at least about 25 contiguousnucleic acids or even at least about 50 contiguous nucleic acids fromsuch proteins). In some preferred embodiments, the amino acid encodes anMCV protein comprising at least about 4 or about 5 or at least about 10contiguous amino acids of the amino-terminal 258 sequence of an MCV T-1polypeptide (one example of which is set forth at SEQ ID NO:12). Ofcourse, the nucleic acid can encode a polypeptide comprising themajority of contiguous amino acids from such proteins, such as all orsubstantially all of such MCV proteins.

For embodiments in which expression of the inventive nucleic acid isdesire, the nucleic acid molecule can be placed into a suitable geneticcontext to promote expression. Thus, in one embodiment, the inventionprovides a composition of matter comprising an expression cassettecomprising a nucleic acid as herein described in operable linkage to asecond nucleic acid having an expression control sequence. An“expression control sequence” is any nucleic acid sequence thatpromotes, enhances, or controls expression (typically and preferablytranscription) of another nucleic acid sequence. Suitable expressioncontrol sequences include constitutive promoters, inducible promoters,repressible promoters, and enhancers. Examples of suitable promotersinclude the human cytomegalovirus (hCMV) promoters, such as the hCMVimmediate-early promoter (hCMV IEp), promoters derived from humanimmunodeficiency virus (HIV), such as the HIV long terminal repeatpromoter, the phosphoglycerate kinase (PGK) promoter, Rous sarcoma virus(RSV) promoters, such as the RSV long terminal repeat, mouse mammarytumor virus (MMTV) promoters, or the herpes thymidine kinase promoter,promoters derived from SV40 or Epstein Barr virus, and the like.

The expression cassette can be placed within a larger nucleic acid andcan include other elements (e.g., sequences for controlling replication,polyadenylation sequences, restriction endonuclease cleavage cites, IRESsites, other expression cassettes (such as encoding proteins conferringresistance to a toxin or other selectable marker) and the like. Theexpression cassette can be constructed by any suitable methodology,which is known to ordinary skill in the art. For example, apolynucleotide encoding an MCV protein as herein described can beligated within a suitable distance of a promoter, and the entirecassette can be further ligated into a desired plasma backbone.Thereafter the construct can be propagated, further engineered, and/orexpressed within a suitable expression system as desired.

It will be further understood that the inventive polynucleic acid(including expression cassettes) can be incorporated within genetransfer vector. Such a vector can facilitate transfer of an expressioncassette into a cell, for example. Alternatively, the vector canfacilitate transfer of an interfering oligonucleotide or siRNA into acell, in conjunction, for example, with a protocol for inhibition ofviral replication or expression. The inventive polynucleotide can beincorporated into any suitable vector system, such as plasmids, cosmids,YACs, viral vector systems (e.g., adenovectors, HSV vectors, retroviralvectors, etc.), which are known to those of ordinary skill in the art.Also, methods of constructing such vectors (e.g., via recombinant DNAtechnology) and of growing and propagating such vectors (e.g., usingsuitable host cells) are known to those of ordinary skill in the art.

Another embodiment of the present invention provides isolated orsubstantially purified MCV proteins and polypeptides. The isolated andsubstantially purified proteins and polypeptides of the presentinvention can be employed to develop antibodies, or as reagents indiagnostic assays. Examples of some such polypeptides include SEQ IDNOs: 6, 8, 10, 12, 14, 16, 18, 20, and that encoded any open readingframe (ORF) of SEQ ID NOs: 1-4. ORFs of SEQ ID NO:1 are represented intable A, and encoded proteins are set forth in FIG. 8, but putative ORFsof SEQ ID NOs: 2-4 and their encoded proteins can be deduced by those ofordinary skill in the art. Typically, the inventive polypeptide includesat least about 5 contiguous nucleic acid residues of an MCV protein, andoften substantially more contiguous nucleic acids from such proteins(e.g., at least about 10 contiguous nucleic acids or at least about 25contiguous nucleic acids or even at least about 50 contiguous nucleicacids from such proteins). In some preferred embodiments, the inventiveprotein comprises at least about 5 or at least about 10 contiguous aminoacids of the amino-terminal 258 sequence of an MCV T-1 polypeptide (oneexample of which is set forth at SEQ ID NO:12). Of course, thepolypeptide can comprise the majority of contiguous amino acids fromsuch proteins, such as all or substantially all of such MCV proteins.

The proteins and nucleic acids of the present invention are isolated orsubstantially purified in the sense that they are separated fromcellular components or mature MCV virions. Thus, in one example,isolated proteins and nucleic acids can exist in substantially (e.g.,90% or more) purified form away from other proteins, polypeptides,and/or nucleic acids. However, it is possible for the inventive proteinsand polypeptides to be present in a combination other than found innatural cellular infection or as a mature virion. Thus, for example, theinventive proteins and polypeptides can be present in an artificialvirus-like particle (VLP).

The proteins/polypeptides and nucleic acids of the present invention canbe produced by standard technologies. For example, particularly withshorter sequences, the inventive proteins/polypeptides and nucleic acidscan be produced by solid-state synthesis. However, it will be understoodthan an efficient method of synthesis involves recombinant DNAtechnology coupled with (in the case of polypeptides) in vitro or invivo translation technology. In many aspects, it is preferable for theinventive polypeptides and proteins to be synthesized using a eukaryoticsynthesis system (e.g., CHO cells), to achieve desirable folding of theamino acid chain and desirable glycosylation patterns.

As noted herein, the MCV proteins and polypeptides can be employed toproduce antibody molecules, which are useful regents for diagnosticassays and potential therapeutic agents. Thus, in another aspect, thepresent invention provides an antibody preparation containing antibodymolecules directed against MCV proteins, polypeptides, and virions. Theantibody molecule that binds specifically to a polypeptide consistingessentially or comprising an amino acid sequence selected from the groupof sequences consisting of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, andany open reading frame (ORF) of SEQ ID NOs: 1-4 or otherwise to an MCVvirion.

The antibody molecules of the present invention typically areimmunoglobulins, and can be of any isotype (e.g., IgA, IgE, IgG, IgI,IgM, and the like) or an active fragment that retains specific bindingactivity (such as Fab fragments and the like). Both polyclonalpreparations and monoclonal antibodies can be prepared by standardtechniques. Following production, the antibody molecules can be isolatedand/or substantially purified, and the invention provides suchantibodies in isolated or substantially purified form.

While not wishing to be bound by theory, it is believed that the T1antigen ORF acquires a mutation during integration into the host cellchromosome that may be responsible for carcinogenesis. The same mutationresults in a truncation of the encoded T1 protein. Thus, it is sometimesdesirable for the antibody to bind specifically to the N-terminalportion of the MCV T-1 protein (e.g., to one or more epitopes containedwithin the N-terminal-most 258 amino acids or so of the MCV T-1 protein.In other embodiments, it is desirable for the antibody molecule to bindspecifically to MCV Large T polypeptide (T-1) without specificallybinding to the MCV Small T polypeptide (T-2). For diagnostic assays, italso is desirable for the inventive antibodies not to bind specificallyto proteins or polypeptides of other polyomaviruses, such as SV-40 LargeT antigen.

In another aspect, the invention provides compositions, which includethe inventive nucleic acid, polypeptide, and/or antibody. In oneembodiment, such a composition can include the inventive nucleic acid,polypeptide, and/or antibody in lyophilized form. Of course, if desired,such a lyophilized composition can include a lyoprotectant, such assucrose or other agent known to those of ordinary skill in the art.Also, the invention provides a composition comprising the inventivenucleic acid, polypeptide, and/or antibody and a carrier, diluent, orbuffer. Such carriers, diluents, and buffers are known to persons ofordinary skill in the art.

In another aspect, the invention provides a virus-like particle (LVP)comprising one or more polypeptides MCV polypeptides selected from thegroup of polypeptides consisting of VP1, VP2, and VP3. Such particlescan be produced by expressing one or more of the VP1, VP2, and/or VP3proteins in a suitable cell. The protein(s) thereafter will assemble toform VLPs, which can be purified from the producing cells by methodssuch as employed with purifying VLPs of other polyomaviruses.

While not wishing to be bound by any particular theory, it is believedthat MCV VLPs form more efficiently if they comprise VP1 molecules froma MCC strain that has consensus-like polyomavirus VP1 sequences at288(Asp), 316(Arg) and/or 366(Asp) (See FIG. 22). In this respect, ithas been observed that the MCV 350 strain does not form VLPs asefficiently as the MCV 339 strain (MCV 350 has differences at 288(His),316(11e) and 366(Asn)). Also, the presence of MCV339-like residues185(Gln) and 422(Glu) might also be important for efficient formation ofVLPs, although these positions are not broadly conserved amongpolyomaviruses.

The VLPs can be used as carriers for foreign DNA, for example, tofacilitate transfection of cells. Thus, the invention provides acomposition of matter comprising a MCV VLP and a non-MCV nucleic acid(e.g., DNA). Such compositions can be made by exposing a VPL to thenon-MCV nucleic acid under conditions suitable for the VLP to bind thenucleic acid. The inventive VLPs, and their use as vectors, can beaccomplished by methods such as are known in the art in connection toother polyomaviruses (see, e.g., Goldmann et al., J Virol Methods. 2000October; 90(1):85-90, Goldmann et al., J. Virol. 1999 May; 73(5):4465-9,Kosukegawa et al., Biochim Biophys Acta. 1996 May 21; 1290(1):37-45,Lundstig et al., Adv Exp Med. Biol. 2006; 577:96-101, Tegerstedt et al.,Anticancer Res. 2005 July-August; 25(4):2601-8, Tegerstedt et al.,Cancer Immunol Immunother. 2007 September; 56(9):1335-44, Viscidi etal., Adv Exp Med. Biol. 2006; 577:73-84, Viscidi et al., Clin Diagn LabImmunol. 2003 March; 10(2):278-85, Yokoyama et al., J Biochem (Tokyo).2007 February; 141(2):279-86, and Zielonka et al., Virus Res. 2006September; 120(1-2):128-37).

In another aspect, the invention provides a pharmaceutical preparationcomprising a composition including the inventive nucleic acid, protein,antibody, and/or VLP and one or more pharmaceutically-acceptableexcipient. Suitable preparations can be formulated for delivery by oral,nasal, transdennal, parenteral, or other routes by standard methodology.In this respect, the excipient can include any suitable excipient (e.g.,lubricant, diluent, buffer, surfactant, co-solvent, glidant, etc.) knownto those of ordinary skill in the art of pharmaceutical compounding(see, e.g., “Handbook of Pharmaceutical Excipients” (PharmaceuticalPress), Rowe et al., 5^(th) Ed. (2006)).

In another embodiment, the invention provides a method of assaying forMCV exposure in a patient, which can be used to assess past exposure,primary infection, or possibly an MCV-associated cancer in the patient.In accordance with this method, a tissue or fluid sample is obtainedfrom the patient. The sample can be, for example, tissue biopsy, blood,plasma, urine, or other fluid. The sample is then assayed for thepresence of one or more MCV molecule(s). Such molecules can be MCV DNA,an MCV polypeptide, or an antibody that binds specifically to an MCVpolypeptide or VLP. The assay for DNA can be facilitated by Northern orSouthern hybridization or PCR. Assaying for an MCV polypeptide, or anantibody that binds specifically to an MCV polypeptide can befacilitated using common immunohistochemical methods. In any event, apositive test for the presence of the MCV molecule within the sample isindicative of exposure of the patient to MCV. Where the test isconducted on tissue obtained from a tumor, the test can facilitatediagnosis of a carcinoma in the patient. Such assays can be used todiagnose patients with MCV-induced cancers or predict which individualsare at greater risk of developing MCV-induced cancers. It is possiblethat high-level MCV infection causes non-cancer disease symptoms. If so,VLPs might be used as a diagnostic tool for primary MCV disease.

A preferred assay for MCV exposure in a patient is an ELISA, in whichthe sample from the patient is exposed to one or more purified MCVpolypeptides, such as VLPs containing VP1, VP2, and/or VP3. Such assayscan be facilitated by high-throughput screening methods employingmulti-well places. In this sense, a multi-well place can be coated withMCV protein or VLPs by standard methods, and the invention provides amulti-well (e.g., 96 well) plate coated with MCV protein and/or VLPs.

Another type of assay (a neutralization assay) is facilitated byinfectious VLPs. In accordance with such an assay, MCV VLPs are producedsuch that they encapsulate a reporter construct (e.g., alkalinephosphatase or Gaussia luciferase). It will be observed, that when suchVLPs infect cells, the reporter is expressed in the cells and can bereadily detected. However, upon exposure of the VLPs to neutralizingantibodies that target MCV prior to exposure to the cells, the titer ofVLPs is substantially reduced, leading to the infection of fewerinfected cells (and fewer cells expressing the reporter). Theneutralization assay can be about 40-fold more sensitive than ELISA fordetection of MCV sero-responses. Accordingly, the assay involvesproducing VLPs that contain a reporter construct, exposing the VLPs to asample, and then exposing the preparation to cells and assaying forexpression of the reporter within the cells. Reduction of reporterexpression in comparison to a control indicates the presence ofneutralizing antibodies in the sample. In this context, the sample canbe obtained from a patient (such as described herein) or a sample of acandidate antibody for clinical use. In this sense, the neutralizingassay can be employed clinically to ascertain patients havingimmunoreactivity to MCV, or it can be alternatively employed to screenfor potential therapeutically-relevant agents targeting MCV (such asimmunoglobulins).

In another embodiment, the invention provides a method of identifying anagent that attenuates MCV infection. In this context, attenuation caninvolve the reduction of likelihood of infection, or reduction inmagnitude. In some applications, the reduction can amount to completeprophylaxis. In accordance with this method, target DNA is exposed to anMCV protein (e.g., VP1, VP2, VP3, T-1, T-2, T-3, T-4, and T-5). Thetarget DNA should include a sequence to which the MCV protein canspecifically bind relative a negative control DNA. The assay isconducted in the presence of a test agent, which is a putative agentunder investigation to assess whether it can attenuate the MCVinfection. Thus, the MCV protein and the target DNA are exposed to eachother under conditions which, except for the test substance, aresuitable for the MCV protein and target DNA to bind. It will beunderstood that, as a result of this assay, the ability of the testsubstance to attenuate binding of the MCV protein to the target DNAidentifies the test substance as a candidate agent for use as ananti-MCV therapeutic agent. An example of this type of assay is agel-shift assay, which is known to those of ordinary skill in the art.Also, while the test agent can be identified as a candidate MCVtherapeutic agent by this method, other tests likely will be needed toassess whether the agent is safe and effective for clinical use.

In another embodiment, the invention provides a method of identifying anagent that attenuates MCV infection by employing triplex DNA technology.In accordance with this method, a test agent is exposed to MCV DNA,wherein the ability of the test substance to promote the formation oftriplex structure within the MCV DNA identifies the test substance as acandidate agent for use as an anti-MCV therapeutic agent. The promotionof triplex DNA can be assessed by standard methods (see, e.g., Havre etal., J. Vivol. 1993 December; 67(12):7324-3). While the test agent canbe identified as a candidate MCV therapeutic agent by this method, othertests likely will be needed to assess whether the agent is safe andeffective for clinical use.

In other aspects, the invention involves prophylactic and therapeuticmethods against MCV diseases. In this context, the MCV disease can beprimary MCV infection or a carcinoma (such as Merkel cell carcinoma,small cell lung carcinoma, or other carcinoma associated with MCVinfection). For example, the invention provides a method of vaccinatinga patient against an MCV disease. In accordance with this method, apatient is vaccinated with MCC DNA and/or a MCV polypeptide underconditions suitable for the patient to generate an immune response tothe MCV DNA and/or MCC polypeptide. A preferred agent for serving as thevaccine is a polypeptide comprising at least 10, and preferably at leastthe majority of, contiguous amino acids from the N terminus of the MCVT1 protein, particularly contiguous amino acids from among theN-terminal approximately 258 amino acids (see SEQ ID NO:12). Anotherpreferred agent is a VLP as herein described. Indeed, rabbits and miceimmunized with MCV can exhibit very high anti-MCV antibody responses,with 50% neutralizing titers in the million-fold dilution range. It willbe understood that MCV VLPs could be combined with other viral subunitvaccines such as the current vaccines against hepatitis B virus andhuman papillomavirus, for combined vaccination protocols.

In another aspect, the invention provides a method for treating apatient suffering from an MCV disease involving adoptive immunotherapy.In accordance with this method, a population of T lymphocytes is firstobtained from the patient. Thereafter, the population of T lymphocytesis exposed ex vivo to an MCV polypeptide, including a VLP (such asdescribed herein) under conditions suitable to activate and expand thepopulation of T lymphocytes. For example, the T lymphocytes can beexposed to cells in vitro, which express an MCV polypeptide (e.g.,having been transfected with an expression cassette encoding the MCVpolypeptide). A preferred MCV polypeptide includes at least 10, andpreferably at least the majority of, contiguous amino acids from the Nterminus of the MCV T1 protein, particularly contiguous amino acids fromamong the N-terminal approximately 258 amino acids (see SEQ ID NO:12).In other aspects, the method can be practices using standard techniques(see, e.g., June, J. Clin. Invest., 117(6) 1466-76 (2007)). After theyhave been activated, at least some of the T lymphocytes arere-introduced into the patient. Such a method can attenuate the severityof the MCV disease within the patient. It should be understood that themethod need not eradicate the MCV disease within the patient to beeffective as a therapy. The method can be deemed effective if it lessenssymptoms, improves prognosis, or augments other modes of therapy if usedadjunctively.

It is believed that the newly-discovered MCV should respond to agentsthat interferes with the replication of other polyomaviruses. Thus, theinvention provides a method of treating an MCV disease by administeringsuch an agent to a patient suffering from an MCV disease. As noted, theMCV disease can be primary MCV infection, Merkel cell carcinoma, smallcell lung carcinoma, or another carcinoma that is caused by MCV. It isbelieved that the administration of some such agents can attenuate theseverity of the MCV disease within the patient. Examples of such agentsare cidofovir and vidarabine, and other agents that interfere withpolyomavirus replication known to those of ordinary skill may be usefulin treating such conditions as well. Additional agents includeinterferons and mTOR inhibitors (e.g., sirolimus and tacrolimus).

It will be understood that the diagnostic therapeutic methods describedherein to be performed on a patient can include human patients as wellas animals. In this respect, the diagnostic and therapeutic methods canbe performed in the veterinary context, i.e., on domestic animals,particularly mammals (e.g., dogs, cats, etc.) oragriculturally-important animals (e.g., horses, cows, sheep, goats,etc.) or animals of zoological importance (apes, such as gorillas,chimpanzees, and orangutans, large cats, such as lions, tigers,panthers, etc., antelopes, gazelles, and others).

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope. In summary,they support the possibility that MCV plays a role in MCC tumorigenesis:

1. MCV is integrated into the tumor cell genome. Precisely identicalintegration sites (confirmed by sequencing) occur in a metastasis(MCC348) and its primary tumor (MCC347). The most likely explanation forthis is that the metastatic tumor arose from a single tumor cell alreadyhaving the virus integration allele.

2. There is an association between MCV and Merkel cell carcinoma.Examination of two different groups of MCC tumors finds themstatistically more likely to be positive for virus than control tissues.In our initial analysis, MCV was present in MCC 43-fold more commonlythan a convenience sample of tissues from different body sites fromdifferent persons. Virus is found only in classic MCC cell linessuggesting that it may be associated with the classic but not variantform of disease. These analyses are based on three independent PCRprimers in a blinded and randomized analysis using stringent PCRsegregation precautions, and confirmed through two additional qPCRassays.

3. Southern blots can detect MCV in tumors. Direct Southern blottingconfirms presence of abundant genome in 8 of 12 tumors and inMCV-positive cell lines. This data indicates that MCV genome is presentat sufficient levels to play a biological role in these tumors. Similardata is not available for other polyomaviruses in human cancer. Thisdata is experimentally independent from PCR-based studies and confirmsthem, demonstrating that the association is not caused by PCRcontamination.

4. MCV is monoclonally integrated into MCC genome in 6 of the 8MCV-positive tumors. Cellular monoclonality is also shown for two tumors(MCC347 and its metastasis MCC348) in which the integration site hasbeen mapped. This suggests MCV infected cells prior to developing intoMCC tumors that subsequently underwent monoclonal expansion. This is notconsistent with MCV being a passenger virus that secondarily infects MCCtumors.

5. MCV T antigens are expressed in tumor cells. DTS and RACE studiesdemonstrate viral T antigen gene expression in tumors infected with MCV.Mutations in the T antigen oncoprotein are consistent with animal modelsof polyomavirus-induced tumors and are not found in non MCC tissuespositive for MCV.

Example 1

This example demonstrates the identification of a previously-unknownpolyomavirus and its integration in Merkel cell carcinoma.

We performed DTS on MCC tissue mRNA and identified T antigen transcriptsfrom a previously undescribed polyomavirus. This virus is closelyrelated to the murine polyoma virus group that includes primate virusessuch as the lymphotrophic African green monkey (AGM) polyomavirus (20),and is more distantly related to all known human polyomavirusesincluding WUV and KIV. The virus is somatically and monoclonallyintegrated into the human genome, suggesting that it was present priorto tumor cell clonal expansion.

Methods and Materials

Human Tissue Sample Testing:

Human Merkel cell carcinoma tissues were obtained from the CooperativeHuman Tissue Network as frozen excess biopsy samples (SupplementaryTable 1). All MCC tumors except MCC352 were reconfirmed in ourlaboratory by H&E and with cytokeratin 20 immunostaining. All MCCtissues except MCC350 were positive for cytokeratin 20. MC350 is MCCmetastatic to a lymph node and due to sampling issues we were unable toidentify MCC tumor cells the portion of tissue taken for ourexamination. We relied on the original pathology report as evidence forMCC. Excess surgical tissues used as controls were collected as aconsecutive series of anonymized pathology collections from a singleoperating day (Supplementary Table 1). Confidentiality issues limitedthe availability of diagnosis for these tissues. Four cases (MCC347,MCC337, MCC343, and MCC346) from 4 men ranging in age from 38 to 84years were used for DTS.

Generation of cDNA Library for Pyrosequencing:

Total RNA was extracted from MCC tissues using RNEASY MIDI kit (Qiagen,Alameda, Calif.) and treated with DNase I (Ambion, Austin, Tex.) toremove genomic DNA. Integrity of tissue RNAs was analyzed by the AGILENT2100 bioanalyzer (Quantum Analytics, Foster City, Calif.) using the RNA6000 Nano reagent kit. mRNA was purified with DYNABEADS mRNApurification Kit (Invitrogen). Double strand cDNA was synthesized witholigo (dT) primer using the SUPERSCIRPT Double-strand cDNA Synthesis kit(Invitrogen). Five microgram of MCC cDNA was used for pyrosequencingafter confirming cDNA quality on an AGILENT bioanalyzer (QuantumAnalytics) at 454 Life Sciences (Roche). The cDNA sample wasfractionated into small fragments (300-500 bp) and blunted for ligationof two different adaptors at both ends. These two adaptors provideunique priming sequences for both amplification and sequencing, formingthe basis of the single-strand template library for pyrosequencingaccordingly. Finally, by GENOME SEQUENCER GS20 system (RocheDiagnostic), large scale sequencing was performed on two cDNA librariesfrom a single (MCC347) and pooled (MCC337, 343 and 346) cases,respectively (M. Margulies et al., Nature 437, 376 (Sep. 15, 2005)).

Digital Transcriptome Subtraction:

The sequences data from large scale library sequencing were firsttrimmed using Lucy (H. H. Chou, M. H. Holmes, Bioinformatics 17, 1093(December, 2001)) with similar Phred scores of 20 or higher (−error 0.010.01), and long read over 50 bp (−m 50). Only high quality sequencesobtained after Lucy trimming were used for further subtraction withSeqClean. First, Poly(A/T), dust (low-complexity), human repeat andprimer adaptors sequences were removed to obtain high fidelity (HiFi)datasets. These HiFi sequences were then aligned against humandatabases, including human Refseq RNA, mitochondrial and assembledchromosomes, and human immunoglobulin variable sequences with a minimumhit length of 30 bp. The remaining sequences were then aligned to onlineGenBank nonredundant (NR) using BLASTX program in netblast package.

RACE Analysis on MCV Transcripts:

Both rapid amplification of 5′ and 3′ cDNA ends (RACE) were performedwith GENERACER Kit (Invitrogen) according to the manufacturer'sinstructions. Primers used for RACE are listed in supplementary table 2.To capture the large T antigen, M1-L primer and M3 were used in 5′RACE.M2-L primer and M4 were used as primers in 3′RACE. To capture the smallT antigen, small.t.R in intron 1 was used in 5′RACE. Small.t.F andsmall.t.F.nest were used in 3′RACE. The PCR fragments were isolated fromagarose gel and extracted with QIAEX II gel extraction Kit (Qiagen), andligated in pCR 2.1 vector (Invitrogen) for DNA sequencing.

Consensus PCR for VP1:

Consensus PCR for the VP1 region of Polyomavirus was previouslydescribed (R. Johne, D. Enderlein, H. Nieper, H. Muller, J Virol 79,3883 (March, 2005)). The genomic DNAs from MCC339, MCC344, MCC347, andMCC350 were subjected to PCR amplification by PLATINUM Taq DNApolymerase (Invitrogen) using two sets of VP1 consensus primers, VP1-1and VP1-2, as in Supplementary Table 2. The cycling conditions for thefirst PCR was 5 min at 95° C., followed by 45 cycles each of 94° C. for30 sec, 46° C. for 1 min and 72° C. for 1 min, and final elongation at72° C. for 10 min. For nested PCR, 4 μl of the first PCR product wasused as the template in a similar reaction at 95° C. for 5 min, 45cycles of 94° C. for 30 sec, 56° C. for 30 min and 72° C. for 30 sec,and 72° C. for 10 min. PCR fragments were recovered from gel, cloned inpCR2.1 cloning vector (Invitrogen) and subjected to nucleotidesequencing. Specific primers (VP1-iF and VP1-iR) for MCV350 VP1 regionwere designed based on the sequencing results.

MCV Genome Sequencing:

The genome was bidirectionally sequenced with at least 3 fold coverage.Successive outward PCR was performed from the 3′ end of the T antigensequence to a conserved VP1 site with primer M6 and VP1_iR, and 5′ endof the T antigen sequences to conserved VP1 site with Primer M5 andVP1_iF. Walking primer set (W1˜W10) was used to sequence the long PCRproduct. Second and third rounds of sequencing used 13 pairs of primers(contig 1˜contig 13) designed to encircle the entire genome. All PCRreactions were performed with HIGH FIDELITY PLATINUM Taq DNA polymerase(Invitrogen). Primers for genome sequencing are listed in SupplementaryTable 3.

Northern Blotting:

Total RNA from 293 cells transfected with pcDNA R339 or R350 containinglarge T genomic region, were extracted by the TRIZOL (Invitrogen).Northern blotting was performed using 5 μg of total RNA for each sample.RNAs were electrophoresed through 1.2% formaldehyde-agarose gels andtransferred onto nitrocellulose membranes (Amersham) with 10×SSC. DNAprobes were generated by random prime labeling of (α32P) dCTP (Amersham)on the P1 MCV DNA fragment which contains the exon-1 sequence of MCV Tantigen (Supplementary Table 4). Hybridization was performed at 42° C.in 5×SSC, 50% formamide, 5×Denhardt's solution, 2% SDS, 10% dextransulfate, and 100 mg/ml of denatured salmon sperm DNA (Stratagene). Finalrinse of the blots were conducted in 2×SSC/0.1% SDS at 60° C. for 30min. RNA ladder marker (Sigma) was used as length control.

MCV Detection by PCR-Southern Blotting:

Genomic DNA was extracted by standard phenol-chloroform technique andthe quality of the DNA was ascertained by PCR with β-actin primers. Onehundred nanograms of genomic DNA was amplified using Taq DNA polymerase(Invitrogen) in a final volume of 50 μl. The cycling condition was 3 minat 94° C., followed by 31 cycles each of 94° C. for 45 sec, 58° C. for30 sec and 72° C. for 45 sec, and final elongation of 15 min at 72° C.Three different primer sets for the T antigen locus (LT1 and LT3) andVP1 gene (VP1) and corresponding primers for the internal probes of Tantigen (M1-M2 and LT5) and VP1 gene (VP1.3) used in Southern blottingare listed in Supplementary Table 4. To avoid potential contamination oftemplate DNA, PCR mixtures were prepared in an isolated room andtemplate DNA was prepared or added in an UV-irradiated clean hood.Recombinant DNA harboring MCV DNA sequence was not amplified at the sametime as tissue samples to avoid cross contamination between PCR samples.Negative controls contained all components except DNA template. Bothcase and control tissue samples were randomized and blinded to thescientist throughout the PCR-Southern testing for MCV positivity.

Genomic DNA Southern Blotting:

Fifteen microgram of each sample, digested overnight with 60 units ofrestriction endonucleases, were separated on 0.7% agarose gels at 80volts. Completion of digestion was checked with ethidium bromidestaining. Gels were transferred onto nitrocellulose membrane (Amersham)with 10×SSC and hybridized overnight with (α32P) dCTP-labelled probe(2.7×107 d.p.m./ml) at 42° C. Membranes were rinsed in 0.2×SSC/0.5% SDSat 60° C. or 72° C. PCR fragments used for the probe synthesis arelisted in Supplementary Table 4. The MCV DNA fragments (LT1, LT2, P1,P3, P6, P9, and P12), which cover 2.5 kb of non-overlapping MCC 350genome, were used for Southern blotting in FIG. 4A. For Southernblotting in FIG. 4B, a probe specific for the intron 1 region of thehuman PTPRG gene (Chr3) was used.

Results and Discussion

Digital Transcriptome Subtraction from Merkel Cell Carcinoma:

To perform DTS, we isolated mRNA from four anonymized MCC tumors fromthe Cooperative Human Tissue Network (21). One case mRNA (MCC347) wasexamined separately, while three case mRNAs were pooled (MCC337, 343 and346) to increase the likelihood for virus discovery (Table 51). Wepyrosequenced 216,599 and 179,135 cDNA sequences (˜150-200 bp) fromthese two libraries, respectively. This allowed us to use allhigh-confidence sequence reads in contrast to our previous DTS analyseswith serial analysis of gene expression (SAGE) tags (15). These 395,734cDNA sequences were trimmed with LUCY stringency equivalent to PHREDscores of 20 or higher (22). Poly(A/T), dust (low-complexity), humanrepeat and primer adaptor sequences were then removed, leaving 382,747sequences to form the HiFi dataset. Of these, 380,352 (99.4%) aligned tohuman Refseq RNA, mitochondrial, assembled chromosomes or immunoglobinNCBI databases. The remaining 2395 sequences were then aligned toGenBank NR using BLASTX.

One transcript (DTS1) from MCC347 aligned over 111 nt. to the DNAbinding domain of human BK polyomavirus T antigen [gi:113204635] with54% identity. The full 201 nt. sequence from this DTS transcript priorto LUCY trimming has highest homology to AGM PyV T antigen [gi:135284](59% identity over 170 nt., 1×e-12). A second DTS transcript (DTS2) fromthe T antigen locus was subsequently identified after alignment ofcandidate HiFi sequences to the full-length viral genome. This fragmentcorresponds to a unique viral T antigen region with low polyomavirushomology. These two sequences define a new human polyomavirus that wecall Merkel cell virus (MCV) because of its close association withMerkel cell carcinoma.

MCV Genome Cloning:

3′-Rapid amplification of cDNA ends (3′-RACE) extended the DTStranscript from case MCC347 to three different cDNAs (FIG. 1B); onetranscript terminated at a poly(A) site in the T antigen sequence andtwo cDNAs read through this poly(A) site to form different lengthfusions with intron 1 of the human receptor tyrosine phosphatase, type G(PTPRG) gene [gi:18860897] at chromosome 3p14.2. Genomic integration atthis site was confirmed by sequencing DNA PCR products from a viral anda PTPRG primer. Identical 3′-RACE cDNA transcripts were independentlyobtained from MCC348, a metastatic lymph node from MCC347, suggestingthat the metastasis was seeded from a clonal tumor cell having the Tantigen-PTPRG fusion.

Viral genome walking was successful on DNA from tumor MCC350 (Table S2)providing the complete closed circular genome (5387 bp, prototype) and asecond genome, MCV339 (5201 bp), was then cloned and sequenced usingspecific primers (Table S3). Both viruses have high homology topolyomavirus T antigen, VP1, VP2/3 and the replication origin sequences(FIG. 7). The principal differences between MCV350 and MCV339 being a201 bp (1994-2184 nt) deletion in T antigen, and a 2 bp (5215-5216 nt)deletion in MCV339 late promoter and a 7 bp deletion (5222-5228 nt.) inthe MCV350 late promoter. Excluding these sites, 41 (0.8%) nucleotidesdiffer between MCV350 and 339. In comparison, 1558 HiFi sequencescomprising 179,301 nucleotides from the MCC347 dataset were alignedwithout gaps to known cellular genes in RefSeq RNA database. Only 130polymorphic nucleotides were found (99.93% concordance), suggesting thathigh mutation rates are not present in this tumor.

Features of the MCV Genome:

MCV has an early gene expression region (196-3080 nt.) containing the Tantigen locus, with large T and small T open reading frames, and a lategene region containing VP1 and VP2/3 open reading frames between 3156and 5118 nt (FIG. 7). The MCV350 replication origin (5360-69 nt) ishighly conserved with seven pentameric T antigen binding sites,including pentanucleotide palindrome and tandem pentanucleotide boxes, ahomopolymeric T tract and semiconserved inverted repeats. Comparison offour MCV genes to those of other polyomaviruses show MCV to be highlydivergent from known human polyomaviruses and SV40 (FIG. 2). MCV hashighest homology to viruses belonging to the MuPyV subgroup and isclosely related to AGM PyV (23).

MCV T Antigen Expression:

To examine MCV T antigen transcription, 3′- and 5′-RACE products weresequenced from MCC 339, 347, 348, 349, 350 and 352 RNAs (FIG. 3). Theseproducts were compared to results of northern blots and RACE productsfrom 293 cells expressing pcDNA-cloned genomic T antigen (48-3695 nt.)fragments from MCV350 and MCV339. Four T antigen spliced products wereidentified in tumors that can be assigned to transcripts expressed fromthe T antigen expression cassettes in 293 cells.

A predicted 2.3 kbase large T transcript (T-1) has near-precise sequencehomology to large T domains from SV40 and other polyomaviruses,including pRB1-binding, DnaJ, Bubl-binding and origin-binding domains aswell as C-terminal helicase/ATPase A, B1 and B2 motifs (24).Surprisingly, stop codons are present in all large T antigens so farsequenced from tumors (MCV339, 347, 348, 349, 350, 352) that willprematurely terminate this protein at different lengths after thepRB1-binding LXCXE motif (FIG. 3), generally deleting origin-binding andhelicase functions. The deletion in MCV339 produces a frameshifted largeT antigen, eliminating expression of the highly-conservedhelicase/ATPase domain. Given the importance of the origin-binding andhelicase domains for replicating episomal virus, these mutations mostlikely arose after viral integration. This transcript forms the fusionto PTPRG in MCC347/348 but is unlikely to generate a fusion protein dueto a stop codon in large T exon 2.

Shorter T antigen proteins are likely to be expressed from other Tantigen transcripts that splice out origin-binding and helicase motifs,but all retain the 5′ crl, DnaJ and LXCXE domains. A small T antigentranscript (T-2) reads through the first splice site. Another Ttranscript (T-3) generates two downstream splice junctions, reminiscentof SV40 17KT (25). Transcripts with obvious, unique homology to rodentvirus middle T sequences were not identified. Genomic integration atdifferent sites within the T antigen locus could also be expected todisrupt full-length gene expression but may still allow proteinexpression from smaller viral transcripts (e.g., T-2, T-3) predicted totarget cell cycle regulatory pathways. Defining the actual T antigenproteins expressed in MCC requires specific antibody panels that do notcurrently exist. But this initial analysis reveals that MCC tumors havean unexpected level of mutational variation affecting T antigen proteinexpression.

Merkel Cell Virus in Merkel Cell Carcinomas:

To determine whether MCV is commonly found in human tissue, 59 controlDNA samples from various body sites were compared to 12 tissues from 10MCC patients (Tables 1, S1). All case and control samples wererandomized and blinded to the scientist testing and scoring two PCRprimer sets in the T antigen locus (LT1 and LT3) and one in the VP1 gene(VP1), followed by Southern blotting with internal probes (Table S4).None of these primer sets amplify plasmid cloned human BK or JC genomicDNA (26, 27).

Of 10 MCC tumors, 7 were positive by PCR using one or more primer setswithout Southern hybridization to amplify detection (Table 1). Oneadditional tumor was positive only after PCR-Southern hybridization. Incomparison, none of the control tissues were positive by PCR alone but 5of 59 (8.5%) samples tested weakly positive after PCR-Southernhybridization, giving an odds ratio of 43 (95% confidence interval 7 to261) for detecting MCV in MCC tumors compared to non MCC tissue samples.Both MCC348 (the metastatic lymph node from tumor MCC347) and MCC338(tumor infiltrating adjacent skin from MCC339) were positive for MCVgenome using multiple PCR primer sets.

In addition to patient samples, common cell lines were tested for thepresence of MCV genome (Table 2). Of four available MCC cell lines, onlyone cell line (MLK-1) grows in suspension culture and is positive forMCV genome by PCR. The three negative cell lines (MCC1, MCC13, MCC26)are adherent “variant” cell lines that have been shown to have distinctgene expression profiles from classical, suspension MCC cells (18). Noneof the nonMCC cells show evidence for MCC infection including COS-7cells containing SV40 genome.

MCV Genomic Integration:

MCV integration can be expected to destroy transmissible virusreplication capacity and thus should be a relatively rare event thatdoes not contribute to viral replication fitness. Integrationnonetheless is frequent in polyomavirus-induced tumors, for example(28), suggesting that this biological accident contributes topolyomavirus tumorigenesis—similar to well-characterized papillomavirusintegration in cervical cancer (29).

Genomic integration can be exploited to examine the origins ofMCV-infected tumor cells. If tumor DNA is digested with single-cutterrestriction endonucleases, such as EcoRI or BamHI, and Southern blottedwith viral sequence probes, four different patterns can be predicted: 1)If virus exists as closed circular episomes or integrated viralconcatemers, then a ˜5.4 kbase band will be present, 2) if MCVintegrates polyclonally—as might to occur during secondary infection ofthe tumor—then diffuse hybridization representing different band sizesis expected, 3) if MCV preferentially integrates at one or a few sites,then tumors will have identical or near identical non-5.4 kbase bandingpatterns, or 4) if MCV integrates at different places in the humangenome prior to tumor clonal expansion, distinct bands of differentsizes will be present (monoclonal viral integration).

Eight of 11 MCC DNA (including MCC348 metastasis from MCC347) showrobust MCV hybridization after BamHI and EcoRI digestion (FIG. 4). Thesesame tumors are also positive by MCV PCR (Table 1). Monoclonal viralintegration is evident with one or both enzymes in six tumors: MCC339,345, 347, 348, 349 and 352 (closed arrows). EcoRI digestion of MCC339,for example, results in two distinct 7.5 and 12.2 kbase bands that canonly arise if MCV is integrated at a single site in the bulk of thetumor mass. MCC344 and 350 bands, in contrast, are consistent withepisomal virus (open arrow), whereas MCC352 has a predominantly episomalor concatenated-integrated pattern but also clear monoclonal integrationbands on BamHI digestion (T antigen sequencing from MCC350, 352 failedto identify a replication-competent T antigen). The banding patterns forMCC347 and its metastasis, MCC348, are identical, consistent with3′-RACE results (FIG. 1B). Of the three Southern blot negative cases,two were negative by PCR-Southern (MCC343 and 346) and the third wasweakly positive with only one PCR primer set (MCC337).

Mapping the human genomic integration site (PTPRG locus on chromosome3p14) for MCC347 and 348 allows us to directly confirm these results.NheI-SacI digestion of MCC347 is predicted to generate a 3.1 kbasefragment from the wild-type allele and a 3.9 kbase fragment from theMCV-integrated allele. As seen in FIG. 4B, the virus-integrated alleleis present in MCC347 and MCC348 DNA, but not control tissues, whenprobed with a flanking human PTPRG sequence probe. Hybridization with aMCV T antigen sequence probe generates the same 3.9 kbase band in MCC347and MCC348, consistent with both cellular and viral monclonality in thistumor. These results together with those in FIG. 4A indicate that MCVinfection and genome integration often occurs prior to clonal expansionof the MCC tumor.

A Potential Role for MCV in MCC: Our results demonstrate an intimateassociation between this new human polyomavirus and Merkel cellcarcinoma. Determination of causality must await confirmation but ourresults suggest that MCV is present in most MCC tumors prior to theirmonoclonal expansion. There are a number of unresolved and interestingquestions. If MCV plays a role in MCC tumorigenesis, we do not knowwhether MCV T antigen expression, insertional mutagenesis or both arecontributing to the tumor phenotype. The PTPRG gene is suspected to be atumor suppressor locus (30) and MCV integration may disrupt itsexpression. Clonality studies (FIG. 4A) indicate that MCV integrationoccurs at other sites and it is possible that dysregulation of the PTPRGpathway is one of several complementing mutations that contribute toMCC.

The potential role for MCV T antigen in tumorigenesis is complex but maylead to critical insights into viral carcinogenesis and immunity.Virus-induced tumors are generally rare biological accidents that do notbenefit viral transmission (31). We have not yet characterized freelyinfectious MCV but mutations in tumor-derived MCV paradoxically preventfull-length large T protein from being expressed. This is likely todestroy origin-binding and helicase activities required for free virusreplication but unlikely to affect integrated virus. Further, Southernblotting for the viral BamHI-EcoRI fragment spanning the T antigen locusreveals deletions and insertions for several viruses that is also likelyto disrupt full large T expression. But these mutations may not affectexpression of smaller T antigens (e.g., T-2 and T-3) that retain domainssuspected to play a role in cancer cell transformation such aspRB1-interaction and DnaJ domains. These findings demonstrate thattumors strongly select against retention of intact MCV large T antigen.Since polyomavirus T antigens provoke robust cytotoxic immune responses(32), these may represent immune escape mutants selected during tumorevolution.

Identifying a new human tumor virus opens diagnostic, therapeutic andprevention possibilities for tumors like MCC that respond poorly tocurrent therapies. In our study, only eight of 10 MCC tumors hadevidence for MCV infection suggesting that MCC may arise from two ormore etiologies. This is supported by gene expression and cell culturestudies that define MCC into classical and variant types (18). Atpresent, we do not know if this accounts for heterogenous MCV infectionin MCC. We also do not know if MCV is a common or uncommon infection ofhumans. Our PCR analysis only shows that MCV is far more common in MCCtissues than an assortment of nonMCC tissues. Addressing the MCCprevalence in human populations requires development of a sensitive andspecific serologic test. Intriguingly, serologic studies led Brade etal. in 1981 to speculate that a virus related to AGM PyV circulates inthe human population (23). Caution is needed in interpreting this,however, since polyomavirus serologic cross-reactivity has been a sourceof confusion (5). PCR contamination has similarly plagued human tumorvirology studies. Direct Southern blotting in our study, however, showsthat MCV genome is present at high copy numbers in most MCC tumorswithout amplification (FIGS. 4A-B).

Sequencing technology and databases have matured so that directhigh-throughput sequencing now can be used to characterize human viralinfections. DTS does not depend on sequence homology and we had no apriori expectation that polyomavirus RNA would be present in MCC. Thisis only practical for hosts, such as humans, in which validated wholegenome sequencing has been accomplished. DTS also has the advantage ofplacing a quantitative upper limit on the abundance of distinguishableviral transcripts when none are found. This does not rule out all formsof infection but it does help to define the possibilities for infectionin a tissue sample.

We chose to study an immune-related tumor based on the concept thatdirect infectious carcinogens express antigenic viral transcripts ineach tumor cell (33). MCC is one of the few tumors significantlyelevated, a 13-fold increase, among AIDS patients in population-baseddatabase cross-matching for AIDS and cancers (33). DTS is less likely tobe useful for tumors caused by chronic inflammation or by viruses thatdo not generate mRNA.

Of the four tumors we chose to initially study, only one was foundretrospectively to have significant MCV virus (Table 1). MCV transcriptsin this tumor (MCC347) are present at 9 transcripts per million orapproximately 2 transcripts per cell. We and others have found that somelatent tumor virus infections retard viral protein synthesis andturnover as a means to evade antigenic peptide processing, withcorrespondingly reduced mRNA transcription (34, 35). Our experience withMCC illustrates that sequencing to <10 transcript per million level onmultiple tissue samples should be used, whenever possible, in searchingfor new human viruses.

TABLE 1 MCV PCR on human tumor (MCC) and control tissues with LT1, LT3and VP1 primers, followed by Southern blot hybridization with internalprobes MCC Cases (n = 10) Patient Tissue ID LT1 LT3 VP1 Summary 1 MCC337+/−* − − +/− 2 MCC338** + + + + MCC339 + + + + 3 MCC343 − − − − 4MCC344 + + + + 5 MCC345 − + − + 6 MCC346 − − − − 7 MCC347 + + − +MCC348*** + + − + 8 MCC349 + + +/− + 9 MCC350 + + + + 10  MCC352ND**** + + + No. of Positives (%) 6/9(66.7) 7/10(70) 7/10(70) 8/10(80)Control Cases (n = 59) Positive Tissues LT1 LT3 VP1 Summary Appendix +/−+/− +/− +/− Appendix − +/− +/− +/− Gall Bladder +/− − − +/− Bowel − +/−+/− +/− Hemorrhoid − − +/− +/− No. of Positives (%) 2/59(3.4) 3/59(5.1)4/59(6.8) 5/59(8.5) *+/−: Signal positive only after Southernhybridization of PCR products. **MCC338, non-tumorous skin tissueadjacent to MCC339. ***MCC348: Metastatic lymph node from MCC347.****ND: Not Determined

TABLE 2 MCV PCR detection in various cell lines Cell Lines Name OriginLT1 LT3 VP1 Summary 293 Human embryonic kidney − − − − COS7SV40-transfected African − − − − green monkey kidney HT1080 Humanfibrosarcoma − − − − MCF7 Human breast cancer − − − − MCC1 MCC − − − −MCC13 MCC − − − − MCC26 MCC − − − − MKL1 MCC + + + +

SUPPLEMENTARY TABLE 1 Clinicopathological data for MCC patients. MCCCases Pa- Tissue Cytoker- tient ID Age Sex Race Phenotype atin 20 1MCC337 84 Male White Malignant + 2 MCC338* 79 Male White Normal + MCC339Malignant + 3 MCC343 79 Male White Malignant + 4 MCC344 57 Mate WhiteMalignant + 5 MCC345 77 Male Black Malignant + 6 MCC346 38 Male UnknownMalignant + 7 MCC347 56 Male White Malignant + MCC348** Malignant + 8MCC349 58 Female White Malignant + 9 MCC350 58 Male White Malignant − 10MCC352 58 Male White Malignant ND*** Control tissue types used in thestudy (MCV PCR+): Colon 5 Small Bowel   3(1) Hemorrhoid   1(1) GallBladder   7(1) Appendix   9(2) Mouth 1 Vein 2 Heart 1 Kidney 1 Skin 9Hernia 2 Hematolymphoid tissues Lymph node 1 Tonsil 5 B cell CLL 1Myeloid hyperplasia 1 Posttransplant lymphoma 1 HIV+ large cell lymphoma1 Miscellaneous tissues Lipoma 1 Fibrous tissue 2 Fistula track 1Meningioma 1 Breast cancer 1 Lung cancer 1 Prostate 1 *MCC338 isnon-tumorous skin tissue adjacent to MCC339 tumor. **MCC348: Metastaticlymph node from MCC347. ***ND: Not Determined

SUPPLEMENTARY TABLE 2 Primers used for the MCV cloning. SEQ ID NamePosition* Purpose Sequence NO M1L 1894-1864 5′-RACETTCTCTTGCAGTAATTTGTAAGGGGACTTAC 46 M3 1848-1827 5′-RACETTTCAGGCATCTTATTCACTCC 47 M2L 1707-1734 3′-RACEAGCAGGCATGCCTGTGAATTAGGATGTA 48 M4 1784-1805 3′-RACETTTTTGCTCTACCTTCTGCACT 49 small.t.R  562-530 5′-RACETAATACAAGCGCACTTAGAATCTCTAAGTTGCT 50 small.t.F  442-473 3′-RACETTTCCTTGGGAAGAATATGGAACTTTAAAGGA 51 small.t.F.nest  496-517 3′-RACEGCTAGATTTTGCAGAGGTCCTG 52 VP1-1F VP1 Consensus PCRCCAGACCCAACTARRAATGARAA 53 VP1-1R VP1 Consensus PCRAACAAGAGACACAAATNTTTCCNCC 54 VP1-2F VP1 Consensus PCRATGAAAATGGGGTTGGCCCNCTNTGYAARG 55 VP1-2R VP1 Consensus PCRCCCTCATAAACCCGAACYTCYTCHACYTG 56 M6 1827-1848 Genome CloningGGAGTGAATAAGATGCCTGAAA 57 VP1iR 3480-3461 Genome CloningATGGGTGAAAAACCCCTACC 58 M5 1796-1770 Genome CloningGGTAGAGCAAAAATTCTTAATAGCAGA 59 VP1iF 3508-3527 Genome CloningCTAGGCAACCCATGAAGAGC 60 *Nucleotide position is based on MCV350 genome

SUPPLEMENTARY TABLE 3 Primers used for genome sequencing SEQ ID NamePosition* Purpose Sequence NO W1  411-4130 Primer walkingACTCTTGCCACACTGTAAGC 61 W2 1290-1272 Primer walking CAGGGGAGGAAAGTGATTC62 W3 4268-4288 Primer walking GGGTAATGCTATCTTCTCCAG 63 W4  946-929Primer walking TATTCGTATGCCTTCCCG 64 W5 4293-4316 Primer walkingCACAGATAATACTTCCACTCCTCC 65 W7 5260-5278 Primer walkingTTATCAGTCAAACTCCGCC 66 W8 5294-5312 Primer walking TCAATGCCAGAAACCCTGC67 W9  166-148 Primer walking AACAGCAGAGGAGCAAATG 68 W10   96-78Primer walking TCTGCCCTTAGATACTGCC 69 contig1f 5344-5363overlapping contigs TTGGCTGCCTAGGTGACTTT 70 contig1r  518-499overlapping contigs CCAGGACCTCTGCAAAATCT 71 contig2f  354-373overlapping contigs GGAATTGAACACCCTTTGGA 72 contig2r  879-860overlapping contigs ATATAGGGGCCTCGTCAACC 73 contig3f  730-749overlapping contigs TGCTTACTGCATCTGCACCT 74 contig3r 1287-1268overlapping contigs GGGAGGAAAGTGATTCATCG 75 contig4f 1132-1151overlapping contigs AGGAACCCACCTCATCCTCT 76 contig4r 1641-1619overlapping contigs AAATGGCAAAACAACTTACTGTT 77 contig5f 1538-1561overlapping contigs AAACAACAGAGAAACTCCTGTTCC 78 contig5r 2088-2069overlapping contigs GAGCCTTGTGAGGTTTGAGG 79 contig6f 1934-1953overlapping contigs AGAGGCCAGCTGTAATTGGA 80 contig6r 2437-2418overlapping contigs GCAGCAAAGCTTGTTTTTCC 81 contig7f 2328-2349overlapping contigs TTTGAAAAGAAGCTGCAGAAAA 82 contig7r 2885-2866overlapping contigs TGTATCAGGCAAGCACCAAA 83 contig8f 2763-2783overlapping contigs CACTTTTTCCCAAAGGCAAAT 84 contig8r 3282-3263overlapping contigs TTACCCAAAGCCCTCTGTTG 85 contig9f 3187-3206overlapping contigs GAGGCCTTTTGAGGTCCTTT 86 contig9r 3687-3667overlapping contigs TCAGACAGGCTCTCAGACTCC 87 contig10f 3599-3618overlapping contigs ATAGAGGGCCCACTCCATTC 88 contig10r 4107-4088overlapping contigs TCTGCCAATGCTAAATGAGG 89 contig11f 3949-3969overlapping contigs CCTGACACAGGAATACCAGCA 90 contig11r 4504-4485overlapping contigs GCAAACTCCAGATTGGCTTC 91 contig12f 4329-4349overlapping contigs TTTTGGAACTGAGGCAACATT 92 contig12r 4829-4810overlapping contigs TAACTGTGGGGGTGAGGTTG 93 contig13f 4765-4784overlapping contigs TACCCACGAAACATCCCTGT 94 contig13r 5386-5367overlapping contigs AGCCTCTGCCAACTTGAAAA 95 *Nucleotide position isbased on MCV350 genome

SUPPLEMENTARY TABLE 4 PCR Primers and Probes used for MCV detection NamePosition* Sense (SEQ ID NO) Antisense (SEQ ID NO)Primers for diagnostic PCR LT1     1514-1953 TACAAGCACTCCACCAAAGC (96)TCCAATTACAGCTGGCCTCT (97) LT3      571-879 TTGTCTCGCCAGCATTGTAG (98)ATATAGGGGCCTCGTCAACC (99) VP1     4137-3786 TTTGCCAGCTTACAGTGTGG (100)TGGATCTAGGCCCTGATTTTT (101)PCR Primers for probes in Northern or Southern hybridizations M1-M2    1711-1889 GGCATGCCTGTGAATTAGGA (102) TTGCAGTAATTTGTAAGGGGACT (103)LT5      253-855 GCTCCTAATTGTTATGGCAACA (104)TGGGAAAGTACACAAAATCTGTCA (105) VP1.3     4107-3599TCTGCCAATGCTAAATGAGG (106) ATAGAGGGCCCACTCCATTC (107) P1     5344-518TTGGCTGCCTAGGTGACTTT (108) CCAGGACCTCTGCAAAATCT (109) P3      730-1287TGCTTACTGCATCTGCACCT (110) GGGAGGAAAGTGATTCATCG (111) P6     1934-2437AGAGGCCAGCTGTAATTGGA (112) GCAGCAAAGCTTGTTTTTCC (113) P9     3187-3687GAGGCCTTTTGAGGTCCTTT (114) TCAGACAGGCTCTCAGACTCC (115) P12     4329-4829TTTTGGAACTGAGGCAACATT (116) TAACTGTGGGGGTGAGGTTG (117) LT2     1054-1428CTGGGTATGGGTCCTTCTCA (118) TGGTGAAGGAGGAGGATCTG (119) Chr.361563308-61563830 TTTCAGACGGAAGCGAAGTT (120) ACCACGATTTGGAAAACAGC (121)*Nucleotide position is based on MCV350 genome ** Nucleotide position isbased on NT_022517.17

The publications referenced in this Example are as follows:

-   1. L. Gross, Proc Soc Exp Biol Med 83, 414 (1953).-   2. K. A. Crandall, M. Perez-Losada, R. G. Christensen, D. A.    McClellan, R. P. Viscidi, Adv Exp Med Biol 577, 46 (2006).-   3. T. Allander et al., J Virol 81, 4130 (2007).-   4. A. M. Gaynor et al., PLoS Pathog 3, e64 (2007).-   5. D. L. Poulin, J. A. DeCaprio, J Clin Oncol 24, 4356 (2006).-   6. J. A. DeCaprio et al., Cell 54, 275 (1988).-   7. D. P. Lane, L. V. Crawford, Nature 278, 261 (1979).-   8. D. I. Linzer, A. J. Levine, Cell 17, 43 (1979).-   9. D. C. Pallas et al., Cell 60, 167 (1990).-   10. M. Cotsiki et al., Proc Natl Acad Sci USA 101, 947 (2004).-   11. D. R. Kaplan, D. C. Pallas, W. Morgan, B. Schaffhausen, T. M.    Roberts, Biochim Biophys Acta 948, 345 (1989).-   12. S. M. Dilworth, Nat Rev Cancer 2, 951 (2002).-   13. D. Ahuja, M. T. Saenz-Robles, J. M. Pipas, Oncogene 24, 7729    (2005).-   14. Y. Chang et al., Science 265, 1865 (1994).-   15. H. Feng et al., J Virol 81, 11332 (2007).-   16. Y. Xu et al., Genomics 81, 329 (2003).-   17. B. Lemos, P. Nghiem, J Invest Dermatol 127, 2100 (2007).-   18. M. Van Gele et al., Oncogene 23, 2732 (2004).-   19. E. A. Engels, M. Frisch, J. J. Goedert, R. J. Biggar, R. W.    Miller, Lancet 359, 497 (2002).-   20. M. Pawlita, A. Clad, H. zur Hausen, Virology 143, 196 (1985).-   21. V. A. LiVolsi et al., Cancer 71, 1391 (1993).-   22. H. H. Chou, M. H. Holmes, Bioinformatics 17, 1093 (2001).-   23. L. Brade, N. Muller-Lantzsch, H. zur Hausen, J Med Virol 6, 301    (1981).-   24. J. M. Pipas, J Virol 66, 3979 (1992).-   25. J. Zerrahn, U. Knippschild, T. Winkler, W. Deppert, Embo J 12,    4739 (1993).-   26. P. M. Howley et al., J Virol 36, 878 (1980).-   27. I. Seif, G. Khoury, R. Dhar, Cell 18, 963 (1979).-   28. D. Hollanderova, H. Raslova, D. Blangy, J. Forstova, M. Berebbi,    Int J Oncol 23, 333 (2003).-   29. M. Durst, A. Kleinheinz, M. Hotz, L. Gissman, Journal of General    Virology (1985).-   30. D. M. Pitterle, E. M. Jolicoeur, G. Bepler, In Vivo 12, 643    (1998).-   31. P. S. Moore, Y. Chang, Annu Rev Microbiol 57, 609 (2003).-   32. T. D. Schell et al., J Virol 73, 5981 (1999).-   33. J. Parsonnet, in Microbes and Malignancy J. Parsonnet, Ed.    (Oxford University Press, New York, 1999) pp. 3-18.-   34. Y. Yin, B. Manoury, R. Fahraeus, Science 301, 1371 (2003).-   35. H. J. Kwun et al., J Virol 81, 8225 (2007).

Example 2

This example demonstrates that MCV is significantly more likely to bepresent in MCC tumors than in control tissues

A second independent set of 8 pathologically-confirmed MCC wererandomized and blindly tested; all 8 (100%) were positive for MCVgenome, assuring reproducibility of our findings. To examine skin MCVinfection, we examined 25 control skin or skin tumor samples from 20HIV-negative and 5 HIV-positive persons without MCC including another 9normal skin samples, Kaposi's sarcoma (n=15) and malignant melanoma(n=1). Four tissues (16%, p<0.001) were positive including one normalskin and three KS lesions, all from HIV negative patients. Thus, MCV issignificantly more likely to be present in MCC tumors (80-100%) than incontrol tissues from patients without MCC (8-16%, p<0.001) (see Table2).

TABLE 3 MCV in MCC and Control Patients by PCR and PCR-Southern TestGroup PCR only PCR-Southern 1. MCC (n = 10)* 7/10 8/10 (80%) 2. MCC (n =8) 8/8   8/8 (100%) 3. Control, various body sites (n = 59) 0/59 4/59(8%)** 4. Control, skin and skin tumor (n = 25) 0/25 4/25 (16%)*** *Twoadditional metatastatic tumors from these patients were also MCVpositive **p < 0.0001 vs. MCC#1; positive tissues included appendix(2/9), gall bladder (1/7), bowel (1/3), hemorrhoid (1/1). Other tissues(all negative) included 9 skin, 6 other GI, 10 lymphoid, 15 othermiscellaneous tissues including organ sites (brain, heart, kidney,lung). ***p < 0.001 vs. MCC#1; positive tissues include KS (3/15),normal skin (1/9). Other negative tissue included malignant melanoma(1).

Example 3

This example demonstrates that MCV has a lymphotropic infection inasymptomatic individuals

Because of MCV's similarity to African green monkey lymphotropicpolyomavirus, we sought to determine if MCV can be detected inperipheral blood from asymptomatic individuals. We have developed qPCRprimers that amplify a region of the T antigen and VP1 genes and can bequantified by comparison to cellular RnasP primers.

Using plasmid dilutions, we find precise linearity over a 4-log DNAdilution for these primers (not shown). We performed a pilot study of 29Multicenter AIDS Cohort Study (MACS) PBMC from HIV-positiveparticipants. Of these 29 PBMC samples, 4 (14%) have >3.5 genomecopies/300 ng DNA indicating asymptomatic infection. Sera from these 4individuals are available in our serum bank and it is evident thatadditional DNA positive individuals can be readily identified by testingadditional individuals with paired PBMC-serum from the MACS repository.A second group of 65 anonymous PBMC collected during routine clinicalstudies (hence HIV status is unknown) shows that 10 (15%) are MCVpositive. Thus, it is likely that MCV is a lymphotropic virus like LPyV.These are initial pilot studies and more detailed and rigorous analysisis needed to determine population rates of infection and possible roleof HIV infection in MCV positivity.

Example 4

Common cell lines (293, COS7, HT1080 and MCF7) were tested and foundnegative for MCV genome. Five MCC cell lines were tested and two arepositive for MCV, providing a renewable source of virus for in vitrostudies. Both MCV-positive cell lines have a classic phenotype whereasthe three negative MCC cells all belong to the variant phenotype,suggesting the possibility that only classic MCC is infected with MCV(Van Gele et al., Oncogene 2004; 23(15):2732-42). This would beanalogous to KSHV in Castleman's disease in which plasmacyticmulticentric Castleman's disease are KSHV-positive but nothyaline-vascular Castleman's disease (Soulier et al., Blood 1995;86:1276-80). qPCR and Southern blotting reveal monoclonal integration ofa single MCV copy into the genome of the MCV-positive cell lines.Additional patient studies are needed to confirm a relationship betweenMCV and classic MCC but this may explain why two MCC tumors were foundto be negative in our PCR analysis (Table 3).

Example 5

This example demonstrates the construction of MCV VLPs.

To produce MCC VLPs, 293TT cells are co-transfected with expressionvectors encoding VP1 and VP2 from either MCV 339 or MCV 350. VLPs arethen extracted from the cells and purified by ultracentrifugationthrough an OPTIPREP density gradient; gradient fractions are collectedat the bottom of the tube (See FIG. 11). Fractions 6, 7, and 8, depictedin FIG. 11 were selected for the presence of nuclease-resistantencapsidated DNA detected using QUANT-IT PICOGREEN dsDNA reagent(Invitrogen).

FIG. 12 demonstrates the production of VLPs for MCV 339 relative to MCV350. The top panel shows an anti-MCV Western blot of 293TT cells aftertransfection with the VP1 expression construct shown, together with anappropriate VP2 expression construct. In the far right lane of theWestern, 5-fold more cell lysate was applied to the gel. The bottompanel shows a SYPRO Ruby-stained SDS-PAGE gel analysis of OPTIPREPgradients used to purify VLPs out of cell lysates. For MPyV and MCV399,2.5 μl each of fractions 6-9 was loaded onto the gel. For MCV350, 12.5μl each of fractions 6-9 was loaded. Fractions were screened for thepresence of encapsidated DNA using PICOGREEN reagent.

These results reveal the production of MCV VLPs from at least one strainof MCV (MCC 339).

Example 6

This example demonstrates the construction of a mouse monoclonalantibody (mAb) specific to the MCV large T (LT) antigen.

Methods

Human Tissue Samples.

DNA samples were obtained from excess clinical specimens. All the DNAsamples were obtained from fresh frozen tissues. For reasons ofconfidentiality, minimal patient identification and demographic data areavailable for most of these specimens. For Merkel cell carcinoma, freshfrozen tumor samples were obtained from the Cooperative Human TissueNetwork (CHTN). An MCC tissue core microarray consisting of 36 MCCspecimens was generated from archival paraffin-embedded tissues from thepathology departments at Hospital Universitari del Mar and the HospitalUniversitari Germans Trias i Pujol, Barcelona, Spain as previouslydescribed (17). Tissue microarrays for lymphoid malignancies and normalcontrols were purchased commercially (US Biomax, Inc.). Genomic DNAsamples from consecutive hematolymphoid tumor tissues were collected andarchived by the late Dr. Anne Matsushima, Columbia University, fromexcess tissue submitted for diagnostic pathology. This was supplementedwith additional hematolymphoid tissues obtained from tissue banks at theUniversity of Pittsburgh Department of Pathology. PBMC specimens wereobtained from two sources: 1) excess samples submitted to the Divisionof Molecular Diagnostics, University of Pittsburgh Medical Center forgenetic screening, and 2) PBMC collectedfrom HIV-positive personsparticipating in Kaposi's sarcoma epidemiologic studies (13). None ofthese study subjects were diagnosed with Merkel cell carcinoma. Allspecimens were tested under University of Pittsburgh InstitutionalReview Board-approved guidelines.

Isolation of genomic DNA. Genomic DNA was extracted using proteinaseK/lysis buffer (0.1 M NaCl, 10 mM Tris-HCl pH 8.0, 25 mM EDTA pH 8.0,SDS 0.5%, 200 μg/ul proteinase K) for up to 3 days at 56° C., followedby phenol-chloroform extraction and ethanol precipitation. DNA amountand quality were determined by spectroscopy followed by PCRamplification for cellular β-actin or RNaseP. Real time quantitative PCR(qPCR). qPCR was performed using primers amplifying the MCV T antigen,TAg (1051 to 1131 nt; forward: 5′-cctctgggtatgggtccttctca-3′ (SEQ IDNO:122), reverse: 5′-atggtgttcgggaggtatatc-3′ (SEQ ID NO:123)) and VP2(4563 to 4472 nt, forward: 5′-agtaccagaggaagaagccaatc-3′ (SEQ IDNO:124), reverse: 5′-ggccttttatcaggagaggctatattaatt-3′ (SEQ ID NO:125))loci with internal TaqMan probes (TAg: 5′-cccaggcttcagactc-3′ (SEQ IDNO:126), VP2: 5′-gcagagttcctc-3′ (SEQ ID NO:127)) labeled with FAM andMGB quencher (Applied Biosystems). For the additional 10 peripheralblood samples with CLL, primers designed against MCV T antigen promoterregion (98 to 184 nt forward: 5′-cccaagggcgggaaactg-3′ (SEQ ID NO:128),reverse: 5′-gcagaaggagtttgcagaaacag-3′ (SEQ ID NO:129)) and internalprobe (5′-ccactccttagtgaggtagctcatttgc-3′ (SEQ ID NO:130)) labeled withFAM and BHQ quencher (Biosearch Technologies) was used. Primers werechosen to maximize specificity to MCV and minimize any cross-reactivitywith other polyomaviruses. Copy numbers were established from standardcurves of Ct values from serial dilutions of known concentrations of MCVDNA originally amplified by PCR using contig 3 andcontig 12 primer setsfor TAg and VP2 detections, respectively (1). Water was used as controlto detect template contamination. No evidence of PCR templatecontamination was observed in the PCR reactions with water control.RNaseP (Applied Biosystems) or β-actin primer-probe mixtures (forward:5′-cactggctcgtgtgacaagg-3′ (SEQ ID NO:131), reverse:5′-cagacctactgtgcgcctacttaa-3′ (SEQ ID NO:132), probe:5′-tggtgtaaagcggccttggagtgtgt-3′ (SEQ ID NO:133)) (BiosearchTechnologies) were used to determine cell genome copy number. qPCRreactions were performed using PRISM 7700 Detection System, PRISM 7900HTFast Real-Time PCR System (Applied Biosystems) and/or SMART CYCLER5RX4Z01 (Cepheid) with TaqMan reagents (UNG (+) TaqMan Universal PCRMaster Mix). All the primers and probes were aliquoted and stored untilin an isolated, clean PCR facility to avoid template DNA contamination.Amplification reactions of all target genes were performed in reactionvolumes of 20 μl with following condition: 50° C. for 2 min, denaturingat 95° C. for 10 min, then denaturing at 95° C. for 15 s followed byannealing and extension at 60° C. for 1 min, 40 cycles. Results wereexpressed as numbers of viral copies per cell calculated from Ct valuesof viral and cellular gene standards (Table 6-1). Cellular viral DNAcopy number below 1.0×10⁻³ per cell was considered to be negative.

Cell lines and Transfection Conditions.

Human embryonic kidney 293 cells (American Type Culture Collection(ATCC)) used for transfection experiment were grown in DMEM mediumsupplemented with 10% fetal calf serum. For protein expression analysis,cells were transfected with expression constructs using LIPOFECTAMINE2000 (Invitrogen) following manufacturer's instructions on 90% confluentcells. Cells were harvested 48 h after transfection for analysis.

Plasmids. To generate the pMCV TAg-EGFP expression constructs, pcDNA6gLT206 encoding wild type full length genomic T antigen (4) was digestedwith Nhe I and Sac II and cloned into pEGFP-N1 (Clonetech) in frame to Cterminus GFP using same restriction sites. LT expression constructs forJCV and BKV were kindly provided by Dr. James DeCaprio (18). SV40 Tantigen cDNA cloned in pCMV vector is described elsewhere (19).

Generation of CM2B4 mAb.

Monoclonal antibody CM2B4 (IgG2b isotype) was generated by standardmethods of immunizing mice with KLH-derivatized SRSRKPSSNASRGA (SEQ IDNO: 134) peptide from the MCV T antigen exon 2 with a C-terminalcysteine (Epitope Recognition Immunoreagent Core facility, University ofAlabama). Immunofluorescence and immunohistochemistry. Forimmunofluorescence staining, cells were spotted on glass slides byCYTOSPIN3 (Shandon), fixed with 10% buffered formalin for 20 min, andpermeabilized with phosphate-buffered saline (PBS) with 0.1% TritonX-100. After blocking with 10% normal donkey serum (JacksonImmunoResearch Laboratories), cells were reacted with CM2B4 (1:100dilution) at 4° C. overnight followed by secondary antibody(Alexa-595-conjugated anti-mouse, 1:1000 Invitrogen) for one hour atroom temperature. Stained cells were mounted in aqueous mediumcontaining DAPI (Vector Laboratories, CA). For immunohistochemicalstaining of paraffin embedded tissues, epitope retrieval was performedusing EDTA antigen retrieval buffer (Dako, Glostrup, Denmark) at 126° C.for 3 min after deparaffinization and hydrogen peroxide treatment. Afterblocking with PROTEIN BLOCK (Dako), samples were reacted to primaryantibody for 30 min at room temperature with dilutions described below.After washing, samples were incubated with Mouse ENVISION Polymer (Dako)for 30 min at room temperature for subsequent deaminobenzidine (DAB)reaction. mAbs used for immunohistochemistry were: CM2B4 (1:10-1:50hybridoma supernatant), CK20 (Dako; 1:50), Chromogranin A (Dako, 1:600),Synaptophysin (Biogenex, San Ramon, Calif., USA; 1:100), and CD56(Novocastra, Newcastle upon Tine, UK; 1:50).

In-Situ Hybridization.

Tissue sections were deparaffinized, dehydrated, incubated at 95° C. for20 min, cooled for 5 min at room temperature and reacted over night at37° C. with JC virus BIOPROBE LABELED PROBE (Enzo Life Sciences) dilutedin hybridization buffer. Excess probe was washed with 2×SSC/0.75% BSAfollowed by PBS. To visualize signal, samples were treated with ABCelite solution (Vector Laboratories, CA) for 30 min, washed twice withPBS and reacted with DAB solution. Samples were counterstained withShandon hematoxylin.

Immunoblotting.

Transfected cells from 6 well plate were lysed in 120 μl of lysis buffer(10 mM Tris-HCl pH8.0, 0.6% SDS) containing proteinase inhibitorcocktail (Roche). 12.5% of lysate was electrophoresed in 10% SDS-PAGE,transferred to nitrocellulose membrane (Amersham). Membranes wereblocked in 5% skim milk for 1 h, reacted with Pab416 (1:10) or CM2B4 mAb(1:10) for overnight at 4° C., followed by anti-mouse IgG-HRP conjugates(Amersham, 1:5000) for 1 h at room temperature. Detection of peroxidaseactivity was performed by WESTERN LIGHTNING PLUS-ECL reagent (PerkinElmer).

Cell Sorting.

5.0×10⁶-10⁷ PBMC were washed with PBS twice, stained with CD3-FITC,CD20-PE and CD14-PC5 (IOTest) (2 μl/10×10⁶ cells) and incubated on icefor 20 minutes. The cells were then washed and resuspended in 400 μl ofPBS containing 8.0 μg/ml DAPI and sorted by the MOFLOW High Speed Sorter(Cytomation). Single antibody stained and unstained cells were used ascontrols for compensation purposes. DNA from the sorted cell fractionswas extracted using QIAAMP BLOOD MINI KIT (Qiagen).

Results

MCV and T Antigen Expression in Merkel Cell Carcinoma Tumors.

We developed a mouse monoclonal antibody (mAb) (CM2B4) to the peptideepitope (SRSRKPSSNASRGA (SEQ ID NO:134)) in exon 2 of the MCV T antigen.This epitope is N-terminal to an LFCDE motif previously found to bindretinoblastoma protein and is likely to be conserved in viruses fromboth tumor and nontumor tissues (4). There was precise nuclearcolocalization of CM2B4 staining with MCV LT-GFP fusion proteinfluorescence when a MCV LT-GFP plasmid was expressed in 293 cells (FIG.9A).

CM2B4 was highly specific for MCV and did not react to T antigens fromJCV or BKV by immunofluorescence (FIG. 9B) or to T antigens from JCV,BKV or SV40 by immunoblotting (FIG. 9C). In contrast, an anti-SV40 Tantigen mAb, PAb416, cross-reacts with T antigens from other SV40-groupviruses including JCV and BKV, but not with MCV T antigen. We examined22 other anti-SV40 T antigen mAbs (Table S1), and none showed reactivityto MCV T antigen on immunoblotting (data not shown). CM2B4 also did notreact to JCV T antigen in a brain biopsy of JCV-infected progressivemultifocal leukoencephalopathy (PML) (FIG. 9D).

The MCV LT protein was detected at 120 kDa on immunoblotting of lysatesfrom cells transfected with the genomic T antigen expression construct(FIG. 9C). An additional 60 kDa band may represent an alternativelyspliced T antigen isoform (4, data not shown). Lysates of the MCVpositive MKL-1 cell line were positive for T antigen expression while Tantigen bands were absent from MCV negative UISO, MCC13, and MCC26 celllines (4) (FIG. 9E).

Immunohistochemical staining of MKL-1 cells showed expression of LTprotein predominantly in a diffuse nuclear pattern (FIG. 10A).Examination of a MCV positive MCC biopsy (MCC349) showed similar strongreactivity with CM2B4 among tumor cells, but not surrounding tissuesincluding the epidermis, adnexal epithelia, endothelial cells, or dermalfibroblasts (FIG. 10B). CK20, a low molecular weight cytokeratin markerfor MCC (9, 10) was present in a characteristic perinuclear dot-likepattern in CM2B4 positive cells (FIGS. 10A and B).

These results were extended by examining a tissue microarray containing30 CK20-positive MCC, 6 CK20-negative but clinically-suspect MCC and 4CK20-negative neuroendocrine control tumors (2 small bowel, one bladderand one lung derived). Of the 30 CK20 positive MCC, 21 (70%) werepositive for LT protein expression. Of the 6 CK20-negative tumorsdiagnosed as MCC, none were positive with CM2B4. These six tumors hadclinical appearances consistent with MCC and expressed neuroendocrinemarkers CD56, synaptophysin, or chromogranin (Table 6-1). These resultssuggest that most CK20-positive MCC express MCV LT in tumor cells.

To screen tissues for MCV infection, we next developed a quantitativereal-time PCR (qPCR) assay to determine the burden of virus infection inMCC tumors (Table 6-2). Ten tumors previously examined by PCR andSouthern blotting (1) were examined with primers designed to amplifyregions of the T antigen and VP2. Seven of these Southern blot positivetumors had an average of 5.2 (range 0.8 to 14) T antigen DNA copies percell. Consistent results were found using VP2 qPCR except for MCC345 andMCC347. This tumor had robust T antigen qPCR positivity (5 copies percell) but minimal amplification of the VP2 gene, which may reflectintegration and loss of this late viral capsid gene region. Thesefindings were confirmed by CM2B4 staining in CK20+ MCCs, which wasconcordant with qPCR results for all cases except MCC344 (Table 6-2).This case showed abundant viral DNA but was negative with CM2B4staining.

PBMC Infection with MCV.

83 whole PBMC DNA samples collected from persons undergoing genetictesting for Factor V Leiden deficiency were tested by qPCR. Thesesamples were collected mainly from adults (average of 60 yrs, range 1-78yrs) with 73 (88%) samples from persons over 18 years of age. None werepositive for viral gene PCR products. Among 21 PBMC collected from adultHIV/AIDS patients without MCC, 2 (9.5%) were positive by either Tantigen (2.8×10⁻³ copies per cell) or VP2 (8.8×10⁻³ copies per cell)primers and one (5%) was positive with both primers (T antigen, 7.9×10⁻³copies per cell; VP2, 6.0×10-3 copies per cell). These levels approachthe technical limit of reproducibility of our assay (>10⁻³ copies percell).

This should not be interpreted as evidence against current infectionamong these participants. PBMC from two MCV-positive MCC patients andfrom three healthy blood donors were sorted into CD20+ (B cell), CD3+ (Tcell), CD14+ (monocyte) and CD20-/CD3-/CD14-(remainder) fractions andtested for T antigen and VP2 DNA. CD20+ B cell fractions from both MCCpatients were positive for both T antigen and VP2 (2.7−9.8×10⁻³ DNAcopies per CD20+ cell). In contrast, CD20+ cell fractions from two ofthree blood donors were only positive for the VP2 locus (1.2−2.1×10⁻³DNA copies per CD20+ cell) but not MCV T antigen. CD3+ T cells from oneMCC patient were positive at lower levels for both viral loci(1.0−2.0×10⁻³ copies per CD3+ cell). Either T antigen or VP2 DNA but notboth were also detected in CD14+ cells from one MCC patient (T antigen:1.2×10⁻³ copies per CD14+ cell), CD20+ cells from two blood donors (VP2:1.2−2.1×10⁻³ copies per CD20+ cell). All other fractions from MCC andblood donor patients were either negative or below the threshold valuefor a positive result. We interpret these results to indicate thatmainly B cell fractions from infected persons can harbor low copy MCVbut dilution of this fraction in whole PBMC reduces MCV below ourreliable threshold for detection.

Survey of Hematolymphoid Malignancies for MCV Infection.

qPCR was performed on DNA from 104 T cell-associated and 161 Bcell-associated malignancies, 19 myeloid disorders and 41 other tumorsincluding Hodgkin lymphoma and post-transplant lymphoproliferativedisorders (Table 6-3). Of these 325 tumors, 7 (2.2%) were positive foreither T antigen or VP2 DNA and two were positive for both. Noconsistent pattern of virus infection was found among thesemalignancies: 1 (3%) of 33 chronic lymphocytic leukemia, 1 (7.1%) of 14non-Hodgkin lymphoma, not otherwise specified (NOS), 2 (3.1%) of 65diffuse large B cell lymphoma, 1 (11%) of 9 marginal zone lymphoma and 1(3.3%) of 30 Hodgkin lymphoma (Table 6-3). Copy numbers for thesepositive hematolymphoid malignancies (Table 6-4), however, were all 2-4logs lower than MCV-positive MCC tumors (Table 6-2).

These results were confirmed by CM2B4 staining of commercial tissuemicroarrays of hematolymphoid tumors. Of 122 B cell lymphomas, 17 T celllymphomas, one myeloid disorder and 2 Hodgkin lymphomas examined, noneshowed evidence for LT protein expression (Table 6-5). Thirty-onehealthy lymphoid control tissues were also negative for MCV T antigen.

MCV and Chronic Lymphocytic Leukemia.

Given the epidemiological relationship between MCC and CLL, we examinedadditional CLL cases for evidence of MCV infection. Ten peripheral bloodsamples with CLL (WBC counts ranging from 13.2×10⁹−84.3×10⁹ cells per L)were harvested and tested for the presence of MCV DNA. One displayed lowMCV positivity VP2 (2.0×10⁻³ copies per cell). Twelve additionalparaffin-embedded biopsies with CLL were examined by CM2B4 staining. AllCLL cases were uniformly negative for MCV T antigen protein expression.

TABLE 6-1 Summary of Merkel cell carcinoma tissue microarray stainingCase number CM2B4 CK20 CD56 Chromogranin Synaptophysin 1 + + + + +2 + + + − + 3 + + + + + 4 + + + + + 5 + + ND^(A) ND ND 6 + + + − ND7 + + + + + 8 + + + + + 9 + + + + + 10 + + + + + 11 + + + + +12 + + + + + 13 + + + + + 14 + + + + + 15 + + + + + 16 + + − + +17 + + + + + 18 + + − + + 19 + + + + + 20 + + + − + 21 + + + + ND 22− + + + + 23 − + + + ND 24 − + + + + 25 − + + + + 26 − + + + + 27 − + +− + 28 − + + + + 29 − + + − + 30 − + + + + 31 − − − − − 32 − − + + + 33− − − − − 34 − − − + + 35 − − + − − 36 − − + − + Control: Neuroendocrinesmall cell carcinoma 37 − − − − + 38 − − + − + 39 − − + + + 40 − − + − +^(A)ND, Not determined.

TABLE 6-2 qPCR detection of MCV genome in MCC MCV genome MCCcopies/cell^(a) Genomic Immunostaining Tissue T Ag VP2 Southern^(b)CM2B4 CK20^(d) MCC337 <10⁻³    0 − − + MCC339    5.2   11.1 + + + MCC343   0    0 − − + MCC344    6.3   13.7 + − + MCC345    4.9 <10⁻³ + + +MCC346 <10⁻³    0 − − + MCC347    1.6    0 + + + MCC349    3.3   8.0 + + + MCC350    0.83    3.0 + NT^(C) NT MCC352   14.3  47.5 + + + ^(a)RNaseP copy number was divided by two to determinecellular equivalent of DNA. ^(b)MCV positivity was previously examinedby Southern blotting (1). ^(c)NT, No paraffin embedded MCC tissues toevaluate. ^(d)CK20 expression was previously examined by immunostaining(1).

TABLE 6-3 qPCR detection of MCV genome in hematolymphoid malignancies.No. MCV positive Hematopathological samples studied No. Tested (% MCVPositive) B cell-associated lymphomas Chronic lymphocytic leukemia 33 1(3.0) Non-Hodgkin lymphoma, NOS 14 1 (7.1) Diffuse large B cell lymphoma65 2 (3.1) Follicular lymphoma 14 0 Acute lymphoblastic leukemia 11 0Primary effusion lymphoma 2 0 Mucosa-associated lymphoid tissue 5 0lymphoma Mantle cell lymphoma 8 0 Marginal zone lymphoma 9 1 (11) Tcell-associated lymphomas Acute lymphoblastic leukemia 10 0 Largegranular lymphocyte leukemia 1 0 Mycosis fungoides 11 0 T cell lymphoma,unspecified 82 1 (1.2) Myeloid disorders Chronic myelogenous leukemia 50 Acute myeloid leukemia 11 0 Myelodysplastic syndrome 3 0 OthersHodgkin lymphoma 30 1 (3.3) Post transplant lymphoproliferative 11 0disorder Total 325 7 (2.2%)

TABLE 6-4 qPCR detection of MCV genome in hematolymphoid malignancies.Copies per cell Hematolymphoid malignancies positive for MCV T Ag VP2Chronic lymphocytic leukemia (#354^(A)) 1.2 × 10⁻² 8.4 × 10⁻³Non-Hodgkin lymphoma (#351) 1.5 × 10⁻³ <10⁻³ Diffuse large B celllymphoma (#229) 1.1 × 10⁻³    0 Diffuse large B cell lymphoma (#500) 3.8× 10⁻³ 1.1 × 10⁻³ Marginal zone lymphoma (#781) 5.8 × 10⁻³    0 T celllymphoma (#18) 3.2 × 10⁻³    0 Hodgkin lymphoma (#86) 1.8 × 10⁻³ 2.9 ×10⁻³ ^(A)Blinded testing number.

TABLE 6-5 MCV LT protein detection in hematolymphoid malignancies B cellmalignancies  0/122 T cell malignancies 0/17 Myeloid disorders 0/1 Hodgkin Lymphoma 02 Normal lymphoid tissues Normal splenic tissue 0/18Normal lymph node 0/13 ^(A)These cases are derived from tissuemicroarray slides #SP482t, #LM801t, #NHL801t from BioMax.

TABLE S1 A list of SV40 T antigen specific monoclonal antibodiesscreened for cross reactivity with MCV T antigen. SV40 T antigenMonoclonal Antibodies Reference(s) PAb101, 108 Gurney, E. G., Tamowski,S., and Deppert, W. 1986. Antigenic binding sites of monoclonalantibodies specific for simian virus 40 large T antigen. J Virol 57:1168-1172. Tack, L. C., Wright, J. H., and Gurney, E. G. 1989.Alterations in the structure of new and old forms of simian virus 40large T antigen (T) defined by age-dependent epitope changes: new T isthe same as ATPase-active T. J Virol 63: 2352-2356. PAb 204, 210, 211,216 Mole, S. E., Gannon, J. V., Ford, M. J., and Lane, D. P. 1987.Structure and function of SV40 large-T antigen. Philos Trans R Soc LondB Biol Sci 317: 455-469. Gannon, J. V., and Lane, D. P. 1990.Interactions between SV40 T antigen and DNA polymerase alpha. New Biol2: 84-92. PAb 405, 409, 407, 416, 419, 423, 430, Harlow, E., Crawford,L. V., Pim, D. C., and Williamson, N. M. 1981. Monoclonal antibodies431, 433, 441, 442 specific for simian virus 40 tumor antigens. J Virol39: 861-869. PAb 602, 603, 605, 606 Mole, S. E., Gannon, J. V., Ford, M.J., and Lane, D. P. 1987. Structure and function of SV40 large-Tantigen. Philos Trans R Soc Lond B Biol Sci 317: 455-469. PAb 901, 902Karjalainen, H. E., Tevethia, M. J., and Tevethia, S. S. 1985.Abrogation of simian virus 40 DNA-mediated transformation of primaryC57BL/6 mouse embryo fibroblasts by exposure to a simian virus40-specific cytotoxic T-lymphocyte clone. J Virol 56: 373-377. Thompson,D. L., Kalderon, D., Smith, A. E., and Tevethia, M. J. 1990.Dissociation of Rb- binding and anchorage-independent growth fromimmortalization and tumorigenicity using SV40 mutants producingN-terminally truncated large T antigens. Virology 178: 15-34. Fu, T. M.,Bonneau, R. H., Epler, M., Tevethia, M. J., Alam, S., Verner, K., andTevethia, S. S. 1996. Induction and persistence of a cytotoxic Tlymphocyte (CTL) response against a herpes simplex virus-specific CTLepitope expressed in a cellular protein. Virology 222: 269-274.

The publications referenced in this Example are as follows:

-   1. Feng, H., Shuda, M., Chang, Y., and Moore, P. S. 2008. Clonal    integration of a polyomavirus in human Merkel cell carcinoma.    Science 319:1096-1100.-   2. Kassem, A., Schopflin, A., Diaz, C., Weyers, W., Stickeler, E.,    Werner, M., and Zur Hausen, A. 2008. Frequent Detection of Merkel    Cell Polyomavirus in Human Merkel Cell Carcinomas and Identification    of a Unique Deletion in the VP1 Gene. Cancer Res 68:5009-5013.-   3. Becker, J. C., Houben, R., Ugurel, S., Trefzer, U., Pfohler, C.,    and Schrama, D. 2008. MC Polyomavirus Is Frequently Present in    Merkel Cell Carcinoma of European Patients. J Invest Dermatol.    advance online publication PMID: 18633441-   4. Shuda, M., Feng, H., Kwun, H. J., Rosen, S. T., Gjoerup, 0.,    Moore, P. S., and Chang, Y. 2008. T antigen mutations are a human    tumor-specific signature for Merkel cell polyomavirus. Proc Natl    Acad Sci USA 105:16272-16277.-   5. Leonard, J. H., Bell, J. R., and Kearsley, J. H. 1993.    Characterization of cell lines established from Merkel-cell    (“small-cell”) carcinoma of the skin. Int J Cancer 55:803-810.-   6. Quaglino, D., Di Leonardo, G., Lalli, G., Pasqualoni, E., Di    Simone, S., Vecchio, L., and Ventura, T. 1997. Association between    chronic lymphocytic leukaemia and secondary tumours: unusual    occurrence of a neuroendocrine (Merkell cell) carcinoma. Eur Rev Med    Pharmacol Sci 1:11-16.-   7. Howard, R. A., Dores, G. M., Curtis, R. E., Anderson, W. F., and    Travis, L. B. 2006. Merkel cell carcinoma and multiple primary    cancers. Cancer Epidemiol Biomarkers Prev 15:1545-1549.-   8. zur Hausen, H., and Gissmann, L. 1979. Lymphotropic papovaviruses    isolated from African green monkey and human cells. Med Microbiol    Immunol 167:137-153.-   9. Moll, R., Schiller, D. L., and Franke, W. W. 1990. Identification    of protein IT of the intestinal cytoskeleton as a novel type I    cytokeratin with unusual properties and expression patterns. J Cell    Biol 111:567-580.-   10. Moll, R., Lowe, A., Laufer, J., and Franke, W. W. 1992.    Cytokeratin 20 in human carcinomas. A new histodiagnostic marker    detected by monoclonal antibodies. Am J Pathol 140:427-447.-   11. Pope, J. H., and Rowe, W. P. 1964. Detection of Specific Antigen    in Sv40-Transformed Cells by Immunofluorescence. J Exp Med    120:121-128.-   12. Diamandopoulos, G. T. 1972. Leukemia, lymphoma, and osteosarcoma    induced in the Syrian golden hamster by simian virus 40. Science    176:173-175.-   13. Moore, P. S., Kingsley, L. A., Holmberg, S. D., Spira, T.,    Gupta, P., Hoover, D. R., Parry, J. P., Conley, L. J., Jaffe, H. W.,    and Chang, Y. 1996. Kaposi's sarcoma-associated herpesvirus    infection prior to onset of Kaposi's sarcoma. AIDS 10:175-180.-   14. Whitby, D., Howard, M. R., Tenant-Flowers, M., Brink, N. S.,    Copas, A., Boshoff, C., Hatziouannou, T., Suggett, F. E. A.,    Aldam, D. M., Denton, A. S., et al. 1995. Detection of Kaposi's    sarcoma-associated herpesvirus (KSHV) in peripheral blood of    HIV-infected individuals predicts progression to Kaposi's sarcoma.    Lancet 364:799-802.-   15. Dorries, K., Vogel, E., Gunther, S., and Czub, S. 1994.    Infection of human polyomaviruses JC and BK in peripheral blood    leukocytes from immunocompetent individuals. Virology 198:59-70.-   16. Stolt, A., Sasnauskas, K., Koskela, P., Lehtinen, M., and    Dillner, J. 2003. Seroepidemiology of the human polyomaviruses. J    Gen Virol 84:1499-1504.-   17. Fernandez-Figueras, M. T., Puig, L., Musulen, E., Gilaberte, M.,    Lerma, E., Serrano, S., Ferrandiz, C., and Ariza, A. 2007.    Expression profiles associated with aggressive behavior in Merkel    cell carcinoma. Mod Pathol 20:90-101.-   18. Poulin, D. L., Kung, A. L., and DeCaprio, J. A. 2004. p53    targets simian virus 40 large T antigen for acetylation by CBP. J    Virol 78:8245-8253.-   19. Campbell, K. S., Mullane, K. P., Aksoy, I. A., Stubdal, H.,    Zalvide, J., Pipas, J. M., Silver, P. A., Roberts, T. M.,    Schaffhausen, B. S., and DeCaprio, J. A. 1997. DnaJ/hsp40 chaperone    domain of SV40 large T antigen promotes efficient viral DNA    replication. Genes Dev 11:1098-1110.

Example 7

This example demonstrates the use of MCV VLPs as reagents in assays forthe detection of MCV infection in human subjects.

Methods

Cases:

22 MCV positive cases were obtained from persons with biopsy-confirmedMCC and qPCR-confirmed MCV infection. Seroprevalence among different agegroups was tested using sera from patients with Langerhans CellHistiocytosis (LCH) (n=151, age of patients: 1 month to 72 years old)were obtained from Dr. Frank Jenkins.

Control:

seroprevalence among adult population was tested using control sera(n=167) were obtained from the New York City Blood Bank (NYCBB) and theColumbia University Blood Bank. All sera were tested for HIV, HCV, HBV,syphilis and were found negative for these infections.

Informed consent from all study participants and IRB approval werereceived in accordance with the guidelines for human experimentations ofthe University of Pittsburgh.

ELISA Assay:

an EIA based on purified VLPs was used to detect presence of specificantibodies in sera samples. Sera were tested using 96-well 2HB Immulonplates (Thermo Scientific), coated with codon-optimized MCV VLP, whichare based on two major capsid proteins, VP1 and VP2. In addition, 12sera samples were tested by CRPV-, BK-, and HPV-VLP ELISA.

For all viruses, 100 μl of purified VLPs at concentration of 1 μg/mlwere added to the wells. After overnight incubation, plates were washedwith PBS and blocked with PBS/0.5% milk for 2 hours at room temperature(see [1] for plate set up).

Serum samples diluted 1:500 were added to 4 wells (2 wells coated withVLP and 2 wells without VLPs) and incubated for 2 hours at roomtemperature. After one-hour reaction with rabbit anti-humanimmunoglobulin G horseradish peroxidase (diluted 1:6000, Dako,Carpenteria, Calif.). Following another washing step3,3′,5,5′-tetramethyl-benzidine (Sigma) substrate was added andincubated for 45 minutes in the dark at room temperature. Reaction wasstopped adding 2N sulfuric acid. Optical density was measured on a MRXplate reader (Dynex Technologies, Chantilly, Va.) at a 405 nm wavelengthwith reference at 620 nm.

Quality Control Testing.

For testing each plate included two test sera standards (MCV high- andmedium-reactive sera), which were tracked over time. These sera werealiquoted and used through out the testing. If results of thesestandards were different by greater than 2 standard deviations from themean optical density, then the results were discarded and the plate wasrepeated. All sera were tested in duplicates, and average ODs for agiven sample were calculated as an average of OD of the wells containingVLPs minus the average of OD of the wells without antigen. Duplicatetests were performed independently, and testing was done in blindedsets.

Competitive ELISA.

12 sera (four high-positive, four-medium, and four negative according toresults of MCV-VLP testing) samples were tested by BK-, and HPV-VLPcompetitive ELISA. Each sera sample was tested by MCV VLP ELISA inserial dilutions: 1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, and1:32000. For each sample, working dilutions were determined to performcompetitive ELISA (FIG. 13).

BK-Competitive ELISA.

200 μl of sera at working dilutions as determined above were incubatedwith 2 ug of BK-VLPs for one hour at room temperature. After incubation,sera with VLPs were added to two wells on the plates, previously coatedwith 100 ng BK-VLPs. Also, two wells were filled with sera withoutBK-VLPs as a control. After two hours of incubation at room temperature,plates were washed three times with PBS and reacted with 100 ul ofrabbit anti-human immunoglobulin G horseradish peroxidase (diluted1:6000, Dako, Carpenteria, Calif.). Following another washing step 100ul of 3,3′,5,5′-tetramethyl-benzidine (Sigma) substrate was added for a45 minute incubation in the dark at room temperature. Reaction wasstopped by adding 100 ul of 2N sulfuric acid.

HPV-Competitive ELISA.

HPV VLP ELISA was performed the same way as BK-competitive ELISA, exceptsera samples were incubated with HPV VLPs and added to the plates,precoated with HPV VLPs.

Peptide Mapping of LT-, MT-, and VP1-Antigens.

In total, 183 peptides (LT), 66 peptides (MT), and 103 peptides (VP1)biotinylated SGSK) 17 mer offset by 5 were synthesized by Mimotop(Clayton Victoria, Australia). An ELISA was performed according to themanufacturer's protocol by using a panel of human serum diluted 1:500.52 samples from LCH patients and 4 positive serum samples were testedfor all three antigens peptides in order to identify specificseroreactive linear epitope for MCV infection.

Results

Peptide ELISA.

Analysis of all 352 peptides studied identified 12 potentialimmunoreactive peptides. An example of the peptide screen by ELISA isshown in FIG. 14. However, when screened against serum from 9 MCCpatients, no specific linear epitope was identified.

MCV VLP ELISA.

Total of 340 serum samples were tested by VLP ELISA. Based on results ofthe test OD value of 0.5 was determined as cutoff. All samples withOD>0.5 were classified as positive.

Among samples derived from MCV-positive individuals 95.5% were positivewith OD>2. Only one sera was negative on the test (4.5%) with OD equalto 0.1. To evaluate prevalence among blood donors two groups of serasamples were tested. In the group of NYCBB (n=105) prevalence was 54.3%.Among Columbia University blood donors (n=62) positive results of thetest were 27.4% of the samples. Absence of demographic data for thesetwo groups does not allow us to interpret this differences inprevalence.

In order to evaluate prevalence of MCV infection in various age groupswe performed testing of 151 serum samples from LCH patients. Accordingto the test results, 29.8% of all samples were positive. Samples weredivided in the 5 groups based on age data: group 1—from 0 to 4 years old(y.o.) (n=29), group 2—from 5 to 9 y.o. (n=32), group 3—from 10 to 14y.o. (n=22), group 4—from 15 to 20 y.o. (n=17), and group 5—from 21 to72 y.o. (n=51).

These serum studies suggest that approximately one-half of normal blooddonors have serum antibodies against MCV, implying that a highproportion of the population has been exposed to MCV. We observed anage-dependent increase in MCV seroprevalence, with about 70% ofindividuals over age 50 showing detectable MCV antibody responses. Theresults of testing are presented in FIG. 15.

VLP Competitive ELISA. BK-Competitive ELISA.

Since cross-reactivity between antibodies to SV40, BKV and JCV VLP hasbeen previously reported, we performed testing for BKV VLP ELISA on 12samples (high-, medium-, and low-positive to MCV VLPs). Results of thetesting demonstrate positive reactivity to BKV (FIG. 16).

To determine if MCV VLP reactivity is due to cross-reaction to BKV, BKcompetitive ELISA was performed. In order to compete out AB to BKV weincubated 100 μl of diluted serum samples with 1 ug of BK VLPs beforeadding to the plates precoated with MCV VLPs. Results of testingdemonstrate that after incubation with BKV VLPs samples were stillreactive to MCV (FIG. 17). Serum samples incubated with BK VLPs showedno reactivity on BK precoated plates. This suggests that MCV reactivityis not due to BKV AB.

CRPV and HPV VLP Competitive ELISA.

12 samples were also tested for CRPV and HPV VLP reactivity. Results ofthe tests are shown in FIG. 18 (A and B).

Prevalence of HPC VLP Positive Reactivity.

As can be seen in FIG. 19, the MCV VLP assay resulted in a nearly 100%positive result for patients who had been diagnosed with MCC. Incontrast, patients with unrelated conditions (lupus, Langerhan's cellhistiocytosis) and from commercial blood sources as well as blood donorstested positive for MCV VLPs at a rate of about 50%. A comparisonbetween the OD from the Elisa assays for MCV+ MCC patients and blooddonors (1:500 dilution) is presented in FIG. 20. The arrow in the rightpanel represents the mean value for the MCV+ MCC patients (2.30 D) less5× the standard deviation for a value of 0.285. While a differentthreshold can be ascertained, these results suggest that an OD valueabove 0.285 might be useful diagnostically.

The publication referenced in this Example are as follows:

-   1. A. S. Laney, J. S. Peters, S. M. Manzi, L. A. Kingsley, Y.    Chang, P. S. Moore. 2006 Use of multiantigen detection algorithm for    diagnosis of Kaposi's sarcoma-associated herpesvirus infection. J.    Clin. Microbiol. 44(10):3734-3741.

Example 8

This example demonstrates the development of a neutralizing assay basedon MCV VLPs.

Neutralization assays were based on the infection of 293TT cells withMCV and MPyV reporter vectors carrying an expression plasmid encodingGaussia luciferase, a secreted reporter protein. Using these MCVreporter vectors, a high-sensitivity MCV neutralization assay wasdeveloped. The neutralization assay is about 40-fold more sensitive thanELISA for detection of MCV sero-responses. 12 MCC patients' MCVsero-responses are, on average, greater in magnitude than responsesfound in 35 normal individuals (p<2.4×10⁻⁵). A small number of normalindividuals were found to exhibit very high MCV sero-responsivenesscomparable to the MCC patients. Validation of the assay is shown in FIG.21 based on titration of MCV-reactive pooled human serum andMPyV-specific rabbit serum. The values for ELISA or neutralizaiton assay(neut) were standardized to calculated maximum optical density (OD) ormaximum relative light units (RLUs), respectively.

Hypothetically, these very high sero-responses may be a record of aprior period of sustained high-level MCV replication. Since MCC tumorstypically do not express the viral capsid genes, it is likely that thisputative episode of high-level virus replication occurred prior todevelopment of the cancer, and may have contributed to the cancer'sdevelopment. Also is expected that the neutralization assay will be lesslikely than the ELISA to detect antibody responses to other humanpolyomavirus types.

Example 9

This example demonstrates the development of a neutralizing assay basedon MCV VLPs.

It is believed that polyomaviruses require two minor capsid proteins,VP2 and VP3 for full infectivity. In previously-characterizedpolyomaviruses, VP3 is an internally-initiated, N-truncated isoform ofVP2. MCVs do not encode a VP2 methionine codon homologous to the codonthat initiates translation of the VP3 ORF of other known polyomaviruses.We therefore produced constructs encoding possible alternative MCV VP3ORFs initiated from VP2 methionine codons 46 or 129. In pilotexperiments, inclusion of expression plasmids encoding MCV VP346enhanced vector infectious titer yield only very modestly, whileinclusion of VP3129 modestly reduced titer yield (data not shown). Theputative MCV VP3 genes were therefore omitted from the MCV vectorproduction scheme. For MPyV, an expression plasmid encoding the standardVP3 ORF was incorporated into the reporter vector production process.

A substantial majority of the VLPs used for the ELISA studies above werefound to contain ˜5 kb fragments of cellular DNA (data not shown). Wetherefore employed a previously-described procedure for enrichingreporter vector stocks for capsids containing reporter plasmids, asopposed to cellular DNA fragments. This strategy resulted in a majorimprovement in particle to infectivity ratios for the reporter vectorstocks (data not shown).

Infection of 293TT cells with an MCV-Gluc reporter vector dose of 400pg/ml (roughly 8 picomolar with respect to VP1 or roughly 100 VLPs percell) resulted in a robust luminescent signal 72 hours after infection.Typical assay conditions resulted in the appearance of roughly 500,000relative light units (RLUs) with a background of roughly 500 RLUs incontrol wells.

To validate the neutralization assay, we tested the ability of pooledhuman sera (PHS) to neutralize the MCV and MPyV Glue vector stocks. ThePHS neutralized the MCV-Glue reporter vector, with 50% neutralization(EC50) occurring at a calculated serum dilution of 1:44,000 (95% Cl1:32,000-1:60,000). In terms of EC50 values, the neutralization assaywas >100-fold more sensitive than the ELISA. This improved sensitivityis presumably due to the 2.500-fold lower dose of virions used in theneutralization assay relative to the ELISA.

In an additional set of assay validation experiments, we found that IgGpurified out of the PHS neutralized the vector with an EC50 of about 90ng/ml). Conversely, PHS stripped of IgG using protein G resinneutralized the MCV reporter vector with an EC50 of only 1:600 (data notshown).

PHS diluted 1:100 failed to neutralize the MPyV reporter vector, whereasan MPyV-specific rabbit serum neutralized the MPyV reporter vector titerby >99% at the same dose. The MPyV-specific rabbit serum only partiallyneutralized the MCV vector at a 1:100 dilution. The results confirm thatMCV and MPyV are not serologically cross-reactive and thatneutralization is due primarily to virus-specific antibodies in varioussera.

We used the MCV neutralization assay to compare serial dilutions of serafrom MCV+ MCC patients (age 14+ years) to sera from a subset of the 48oldest plasma donors (age 47-74 years). We also tested sera from LCHpatients age 47-72 years in the neutralization assay. A small number ofMCC patients whose tumors were found not to contain MCV were alsotested. MCV+ MCC patients displayed very high titer MCV-neutralizingresponses that were not typical among control donors. This differencewas statistically significant. Although the apparent difference betweenthe neutralizing titers of MCV+ and MCV− MCC patients was notstatistically significant, several of the MCV− patients displayedneutralizing titers much lower than titers observed for MCV+individuals, suggesting that a minority of MCC cases are MCV−independent.

88% (42/48) of the plasma donor sera detectably neutralized theinfectivity of the MCV reporter vector at the lowest serum dilutiontested (1:100). In contrast, only half (24/48) of this subset of serascored seropositive in the VLP ELISA. To address this discrepancy, weretested this subset of sera at a more concentrated dilution (1:40) inthe VLP ELISAs. For the re-testing, any serum whose raw OD against MCVVLPs was at least three-fold greater than its raw OD against MPyV VLPswas defined as seropositive. By this standard, 75% (36/48) of the plasmadonor sera displayed MCV-specific ELISA reactivity, in a patterngenerally correlating with neutralizing titers. Taken together, theresults suggest that, in addition the roughly 50% of donors who displayrobust anti-MCV antibody responses, an additional 25% of donor sera haveweak, but detectable, MCV-specific reactivity.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

The invention claimed is:
 1. An isolated or substantially purifiedmurine monoclonal antibody molecule that binds selectively to apolypeptide consisting essentially of an amino acid sequence selectedfrom the group of sequences consisting of SEQ ID NOs: 6, 8, 10, 12, 14,16, 18, 20, and any open reading frame (ORF) of SEQ ID NOs: 1-4.
 2. Theantibody molecule of claim 1, wherein the polypeptide consistsessentially of the amino acid sequence of SEQ ID NO:6.
 3. The antibodymolecule of claim 1, wherein the polypeptide consists of the amino acidsequence of SEQ ID NO:6.
 4. The antibody molecule of claim 1, whereinthe polypeptide consists essentially of the amino acid sequence of SEQID NO:8.
 5. The antibody molecule of claim 1, wherein the polypeptideconsists of the amino acid sequence of SEQ ID NO:8.
 6. The antibodymolecule of claim 1, wherein the polypeptide consists essentially of theamino acid sequence of SEQ ID NO:10.
 7. The antibody molecule of claim1, wherein the polypeptide consists of the amino acid sequence of SEQ IDNO:10.
 8. The antibody molecule of claim 1, wherein the polypeptideconsists essentially of the amino acid sequence of SEQ ID NO:12.
 9. Theantibody molecule of claim 1, wherein the polypeptide consists of theamino acid sequence of SEQ ID NO:12.
 10. The antibody molecule of claim1, wherein the polypeptide consists essentially of the amino acidsequence of SEQ ID NO:14.
 11. The antibody molecule of claim 1, whereinthe polypeptide consists of the amino acid sequence of SEQ ID NO:14. 12.The antibody molecule of claim 1, wherein the polypeptide consistsessentially of the amino acid sequence of SEQ ID NO:16.
 13. The antibodymolecule of claim 1, wherein the polypeptide consists of the amino acidsequence of SEQ ID NO:16.
 14. The antibody molecule of claim 1, whereinthe polypeptide consists essentially of the amino acid sequence of SEQID NO:18.
 15. The antibody molecule of claim 1, wherein the polypeptideconsists of the amino acid sequence of SEQ ID NO:18.
 16. The antibodymolecule of claim 1, wherein the polypeptide consists essentially of theamino acid sequence of SEQ ID NO:20.
 17. The antibody molecule of claim1, wherein the polypeptide consists of the amino acid sequence of SEQ IDNO:20.
 18. The antibody molecule of claim 1, wherein the polypeptideconsists essentially of the amino acid sequence of an ORF of SEQ IDNO:1.
 19. The antibody molecule of claim 1, wherein the polypeptideconsists of the amino acid sequence of an ORF of SEQ ID NO:1.
 20. Theantibody molecule of claim 1, wherein the polypeptide consistsessentially of the amino acid sequence of an ORF of SEQ ID NO:2.
 21. Theantibody molecule of claim 1, wherein the polypeptide consists of theamino acid sequence of an ORF of SEQ ID NO:2.
 22. The antibody moleculeof claim 1, wherein the polypeptide consists essentially of the aminoacid sequence of an ORF of SEQ ID NO:3.
 23. The antibody molecule ofclaim 1, wherein the polypeptide consists of the amino acid sequence ofan ORF of SEQ ID NO:3.
 24. The antibody molecule of claim 1, wherein thepolypeptide consists essentially of the amino acid sequence of an ORF ofSEQ ID NO:4.
 25. The antibody molecule of claim 1, wherein thepolypeptide consists of the amino acid sequence of an ORF of SEQ IDNO:4.